Expression Of Markers Research Articles

Clinicopathological observation and analysis of uveal leiomyoma

Objective: To analyze the clinical and histopathological features of uveal leiomyoma. Methods: A retrospective study was performed on 19 patients with uveal leiomyoma confirmed by histopathological examination in Beijing Tongren Hospital from January 2018 to May 2023. All patients underwent local resection of the tumor. Clinical data of the patients, such as gender, age, clinical manifestations, tumor location, size, and B-scan ultrasound examinations were collected. The pathological morphological characteristics of the tissue (cell type) and the expression of the immune markers, including smooth muscle actin, actin, and neural cell adhesion molecule (CD56), were evaluated. Results: Among the 19 intraocular leiomyoma patients, there were 5 males and 14 females. The average age was (38.9±13.7) years, ranging from 14 to 64 years. The course of the disease ranged from 1 to 36 months with an average of (9.3±8.7) months. The most common clinical manifestation was blurred vision (12/19), followed by decreased vision (4/19), increased IOP (2/19), and visual occlusion (1/19) in patients. The tumor was located in the ciliary body in 10 female and 5 male patients, including 2 patients with the iris involved, and in the choroid in 4 female patients. Two patients with a history of breast fibronenoma had uveal leiomyoma in the ciliary body and the choroid, respectively. One patient with a history of uterine leiomyoma suffered uveal leiomyoma in the ciliary body. B-scan ultrasonography showed that the tumor was a solid and medium-low echoic lesion with a clear boundary in all eyes. We divided these leiomyomas into two histopathological types: spindle cell type (15/19) and round cell type (4/19). Immunohistochemical staining disclosed smooth muscle actin and actin were expressed in all tumor cells (19/19, 100%), and CD56 was positive in 16 of 19 eyes (84.2%). The positive expression rates of estrogen and progesterone were 0/19 and 2/19. During the follow-up of 7 to 71 months after surgery, no tumor metastasis occurred. Only 1 male patient had tumor recurrence. Conclusions: The most common site of uveal leiomyoma is the ciliary body, the major clinical manifestation is blurred vision, and the prognosis is good. Most of the tumors are spindle cell tumors, with myogenic and neurogenic histological characteristics. Although uveal leiomyoma is more frequently found in women of reproductive age, it shows no significant correlation with estrogen and progestational hormones.

DDDR-26. IDENTIFICATION OF MODULATORS OF IDH INHIBITOR SENSITIVITY INIDH-MUTATED DIFFUSE GLIOMAS

Abstract Driver mutations in IDH1 and IDH2 characterize a substantial proportion of lower-grade diffuse gliomas in adults. Mutated IDH molecules cause the accumulation of D-2-hydroxyglutarate (D-2-HG), which perturbs many cellular functions, including metabolism, epigenetic regulation, and the ability to differentiate. This is notably associated with DNA and histone hyper-methylation. Recently, the newly developed IDH inhibitor, vorasidenib, was shown to delay tumor progression and the need for additional treatment in the groundbreaking INDIGO clinical trial. Nevertheless, the responses to vorasidenib were variable between patients, and so far have been established only for less aggressive disease. In addition, it is unclear to what extent vorasidenib can cause tumor regression. This highlights the need to identify molecules and pathways that control IDH inhibitor sensitivity. Towards this goal, we generated a new mouse model that combines Idh1R132H and Trp53 loss-of-function, targeted to oligodendrocyte progenitors. All the mutant mice develop diffuse gliomas that recapitulate the cardinal features of the corresponding human disease. We established multiple cell lines from these tumors, which retained expression of mutant IDH. The cells robustly produced D-2-HG, which was concentration-dependently suppressed by vorasidenib. Accordingly, vorasidenib could lower DNA methylation, and induce the expression of mature glial cell markers. To identify determinants of vorasidenib sensitivity, we performed genome-wide loss-of-function CRISPR/Cas9 functional genomics screens. In those experiments, targeting genes associated with astrocyte differentiation or Notch signaling enhanced cell fitness in the presence of vorasidenib, consistent with the anticipated effects of the drug on inducing cell differentiation. Conversely, vorasidenib-treated cells were more sensitive to the loss of regulators of cell growth and metabolism, and of some epigenetic regulators. These studies point to strategies that may enhance the efficacy of IDH inhibitors for the treatment of IDH mutated gliomas.

Electroacupuncture alleviates neuropathic pain in a rat model of CCD via suppressing P2X3 expression in dorsal root ganglia

Abstract Background Sciatica and low back pain are prevalent clinical types of neuropathic pain that significantly impair patients' quality of life. Conventional therapies often lack effectiveness, making these conditions challenging to treat. Electroacupuncture (EA) is an effective physiotherapy for pain relief. Prior research has demonstrated a relationship between the frequency of neuropathic pain and the analgesic impact of EA stimulation. This work aimed to assess the analgesic effects of EA in a rat model of chronic compression of the dorsal root ganglion (CCD) and to understand the underlying processes. Methods We established a rat CCD model to simulate sciatica and low back pain. EA was applied to rats with CCD at various frequencies (2 Hz, 100 Hz, and 2/100 Hz). The paw withdrawal threshold (PWT) was measured to assess analgesic effects. Additionally, protein levels of the purinergic receptor P2X3 (P2X3) and the expression of nociceptive neuronal markers were analyzed using immunohistochemistry and western blot (WB) techniques. The study also measured levels of proinflammatory cytokines TNF-α and IL-1β in the dorsal root ganglion (DRG). The involvement of P2X3 receptors was further investigated using the P2X3 agonist, α,β-methylene ATP (α,β-meATP). Results CCD rats developed pronounced mechanical allodynia. EA stimulation at all tested frequencies produced analgesic effects, with 2/100 Hz showing superior efficacy compared to 2 Hz and 100 Hz. The expression of P2X3 was increased in ipsilateral DRG of CCD model rats. P2X3 were co-labeled with isolectin B4 (IB4) and transient receptor potential vanilloid (TRPV1), indicating their role in nociception. 2/100 Hz EA treatment significantly reduced mechanical allodynia and inhibited the overexpression of P2X3, TRPV1, substance P (SP), and calcitonin gene-related peptide (CGRP) in the ipsilateral DRG of CCD model rats. Additionally, EA reduced the levels of proinflammatory cytokines TNF-α and IL-1β in the ipsilateral DRG, indicating an anti-inflammatory effect. The P2X3 agonist α,β-me ATP attenuated the analgesic effect of 2/100 Hz EA in CCD rats. The WB and immunofluorescence results consistently demonstrated P2X3 inhibition contributed to the analgesic effects of 2/100 Hz EA on CCD-induced neuropathic pain. Conclusions Our findings suggest that 2/100 Hz EA alleviates neuropathic pain in rats by inhibiting the upregulation of P2X3 receptors in the ipsilateral DRG. This study backs up EA as a viable treatment option for sciatica and low back pain in clinical settings.

EXTH-39. A BENCH-TOP MODEL FOR THE OPTIMIZATION OF ULTRASOUND PARAMETERS FOR SONODYNAMIC THERAPY OF GLIOBLASTOMA

Abstract BACKGROUND In-vitro studies have demonstrated that low frequency sound waves can interact with hemo-porphyrin molecule to induce the production of reactive oxygen species. This biophysical phenomenon can induce tumour cell death in glioblastoma cells in-vitro. The purpose of this study was to evaluate the production of reactive oxygen species and expression of apoptotic markers in glioblastoma cells in response to varying total treatment time and ultrasound power using a bench-top focused ultrasound (FUS) device. METHODS U251 cells were exposed to 5-Aminolevulenic Acid (5-ALA) at varying concentrations for up to 24 hours. The time point with peak proto-porphyrin IX (PPIX) fluorescence was established using fluorescence activated cell sorting (FACS). A transducer utilized at 0.91MHz was placed on an inverted stage with the focus targeted to the bottom of a standard 96-well cell culture plate. Pressure wave delivery was confirmed using a hydrophone. GBM cells were treated with 5-ALA alone, FUS alone, or 5-ALA + FUS and the cells were incubated for 24 hours prior to measuring ROS induction and cell death. ROS was measured using FACS with the CellROX Green Kit for Oxidative Stress Detection and Annexin-V was measured using the Dead Cell Stain Kit. RESULTS Peak fluorescence intensity for PPIX in the U251 cell line was found to be induced through incubation with 5-ALA for 24 hours with a concentration 200mg/mL. The fluorescence intensity of PPIX was found to be 313% greater in the treated group compared to the control group. Peak ROS was observed using a 0.91MHz transducer frequency for a total time of 120s with 6W average power. ROS was 61% and 66% greater in the SDT group compared to the FUS and 5-ALA group respectively. CONCLUSION Using this bench-top set up we successfully induced ROS and apoptosis in U251 cells. Establishing a reliable in-vitro model will allow us to further investigate the effects of other sonication parameters such as burst length.

Hedgehog Pathway Is a Regulator of Stemness in HER2-Positive Trastuzumab-Resistant Breast Cancer

HER2 overexpression occurs in 20–30% of breast cancers and is associated with poor prognosis. Trastuzumab is a standard treatment for HER2-positive breast cancer; however, resistance develops in approximately 50% of patients within a year. The Hedgehog (Hh) signalling pathway, known for its role in maintaining stemness in various cancers, may contribute to trastuzumab resistance in HER2-positive breast cancer. This study aimed to investigate the role of Hedgehog signalling in maintaining stemness and contributing to trastuzumab resistance in HER2-positive breast cancer cell lines. Trastuzumab-resistant HER2-positive breast cancer cell lines, SKBR3 and HCC1954, were developed through continuous trastuzumab exposure. Cells were treated with GANT61 (Hh inhibitor, IC50:10 µM) or SAG21K (Hh activator, IC50:100 nM) for 24 h to evaluate the Hedgehog signalling response. Stemness marker expression (Nanog, Sox2, Bmi1, Oct4) was measured using qRT-PCR. The combination index (CI) of GANT61 with trastuzumab was calculated using CompuSyn software(version 1.0) to identify synergistic doses (CI < 1). The synergistic concentrations’ impact on stemness markers was assessed. Data were analysed using two-way ANOVA and Tukey’s post hoc test (p < 0.05). Trastuzumab-resistant cells exhibited increased Hedgehog signalling activity. Treatment with GANT61 significantly downregulated stemness marker expression, while SAG21K treatment led to their upregulation in both SKBR3-R and HCC1954-R cells. The combination of GANT61 and trastuzumab demonstrated a synergistic effect, markedly reducing the expression of stemness markers. These findings indicate that Hedgehog signalling plays a pivotal role in maintaining stemness in trastuzumab-resistant cells, and that the inhibition of this pathway may prevent tumour progression. Hedgehog signalling is crucial in regulating stemness in trastuzumab-resistant HER2-positive breast cancer. Targeting this pathway could overcome resistance and enhance trastuzumab efficacy. Further studies should explore the clinical potential of Hedgehog inhibitors in combination therapies.

IMMU-36. MULTI-OMIC CHARACTERIZATION OF A SYNGENEIC MOUSE CANCER STEM CELL MODEL OF GBM FOR PRECLINICAL APPLICATIONS

Abstract Glioblastoma (GBM) is resistant to many therapies including immunotherapies. There is an urgent need to explore the role of the microenvironment in resistance to therapy with tailored pre-clinical models. Using multi-omic approaches we describe a syngeneic cancer stem cell mouse model of GBM, with a spontaneous amplification of Igf2. We investigate whether Igf2 influences tumour and microenvironment cells to promote immunosuppression in GBM. Three cell lines were previously established from spontaneous brain tumours in C57Bl/6 Trp53+/-/Nf1+/- mice and maintained under neural stem cell culture conditions. Whole genome sequencing (WGS) was used to determine single nucleotide, copy number, and structural variation, revealing loss of both copies of Trp53 and Nf1 in all three cell lines. However only one cell line, mBT0309, developed tumors when orthotopically allografted. Analysis of genomic alterations and transcriptomics revealed that mBT309 exhibits amplification of the Igf2 loci and overexpression was confirmed at the RNA and protein level. Spatial and single cell transcriptomics showed that Igf2 is overexpressed in mBT0309 allografted tumours and correlated with specific transcriptomic programs. A high-parameter Imaging Mass Cytometry (IMCTM) panel was used for spatial proteomic analysis, to monitor the development of tumours in a time-course experiment. In vitro growth characteristics and stem marker expression in both transcriptomic and spatial IMC data suggest that high levels of Igf2 may regulate GBM stemness features. Analysis of human GBM datasets revealed that Igf2 amplified tumours have reduced CXCL11 expression. These findings suggest that high levels of Igf2 may influence immunosuppression by reducing T-cell recruitment. This syngeneic immunocompetent GBM stem cell model harbouring Igf2 amplification adds to the suite of models to study the GBM microenvironment and its role in immunosuppression and resistance to therapy.

Surrogate immunohistochemistry markers in adult gliomas – Experience in a tertiary care hospital

Objectives: The recent World Health Organization classification has recommended the usage of surrogate immunohistochemical markers for molecular classification of gliomas. However, only a few studies have attempted to study the expression of the entire panel of markers. The current study was undertaken to study the association of recognized surrogate immunohistochemical markers (isocitrate dehydrogenase 1 [IDH-1], alpha-thalassemia mental retardation X-linked [ATRX] and p53) in different histological lineages and grades of adult gliomas. Materials and Methods: This study was conducted on 118 cases of adult gliomas diagnosed on histopathology over a 2-year duration in a tertiary care hospital. The expression of surrogate immunohistochemistry markers (IDH-1, ATRX, and p53) in these cases was studied. Statistical analysis: Descriptive statistical analysis with the Statistical Package for the Social Science system version 17.0. Results: The frequency of IDH-1 positivity was significantly higher in oligodendrogliomas (OG: 76.5%; oligoastrocytoma: 100%) versus astrocytoma (AS) (grades 2 and 3: 48.1%). It was also significantly higher in diffuse gliomas (grades 2 and 3) versus glioblastomas (64% vs. 16.9%). Among IDH-mutant diffuse gliomas, ATRX loss was significantly higher in AS versus OGs (84.6% vs. 7.7%). P53 overexpression correlated significantly with histological subtype (AS 2,3: 55.6% vs. OG: 5.9%). Conclusions: The surrogate immunohistochemical panel of IDH-1, ATRX, and p53 showed significant association with distinct histopathological subtypes and is helpful in molecular stratification. Cut-offs of ≥ 10% nuclear positivity for p53 and 50% loss of nuclear ATRX expression showed a good correlation.

Characterization and Experimental Use of Multiple Myeloma Bone Marrow Endothelial Cells and Progenitors

Multiple myeloma (MM) is a plasma cell malignancy that resides within the bone marrow microenvironment, relying heavily on interactions with its cellular components. Among these, endothelial cells (ECs) play a pivotal role in MM progression and the development of therapeutic resistance. In this study, we analyzed publicly available single-cell RNA sequencing data to identify unique pathway activations distinguishing ECs from MM patients and healthy donors. We developed a novel protocol to isolate and culture endothelial progenitor cells (EPCs) and ECs directly from MM patient bone marrow, demonstrating their ability to promote myeloma cell proliferation. Validation studies confirmed that these MM-derived ECs exhibit angiogenic potential as well as the expression of characteristic endothelial lineage markers. These findings underscore the critical role of bone marrow ECs in the MM tumor microenvironment and highlight potential new therapeutic targets to disrupt MM progression.

Intermittent Fasting Improves Insulin Resistance by Modulating the Gut Microbiota and Bile Acid Metabolism in Diet‐Induced Obesity

ScopeAdipose tissue macrophages (ATMs) are crucial in the pathogenesis of insulin resistance (IR). Intermittent fasting (IF) is an effective intervention for obesity. However, the underlying mechanism by which IF improves IR remains unclear.Methods and resultsMale C57BL/6J mice are fed chow‐diet and high‐fat diet (HFD) for 12 weeks, then is randomized into ad libitum feeding or every other day fasting for 8 weeks. Markers of ATMs and expression of uncoupling protein 1 (UCP‐1) are determined. Gut microbiota and bile acids (BAs) are profiled using 16S rRNA sequencing and targeted metabolomics analysis. Results indicate that IF improves IR in HFD‐induced obesity. IF decreases ATM infiltration, pro‐inflammatory M1 gene expression, and promotes white adipose tissue (WAT) browning by elevating UCP‐1 expression. IF restructures microbiota composition, significantly expanding the abundance of Verrucomicrobia particularly Akkermansia muciniphila, with the decrease of that of Firmicutes. IF increases the level of total BAs and alters the composition of BAs with higher proportion of 12α‐hydroxylated (12α‐OH) BAs. The changes in these BAs are correlated with differential bacteria.ConclusionThe findings indicate that IF improves IR partially mediated by the interplay between restructured gut microbiota and BAs metabolism, which has implications for the dietary management in obesity.

Identifying and optimizing critical process parameters for large-scale manufacturing of iPSC derived insulin-producing β-cells

BackgroundType 1 diabetes, an autoimmune disorder leading to the destruction of pancreatic β-cells, requires lifelong insulin therapy. Islet transplantation offers a promising solution but faces challenges such as limited availability and the need for immunosuppression. Induced pluripotent stem cells (iPSCs) provide a potential alternative source of functional β-cells and have the capability for large-scale production. However, current differentiation protocols, predominantly conducted in hybrid or 2D settings, lack scalability and optimal conditions for suspension culture.MethodsWe examined a range of bioreactor scaleup process parameters and quality target product profiles that might affect the differentiation process. This investigation was conducted using an optimized High Dimensional Design of Experiments (HD-DoE) protocol designed for scalability and implemented in 0.5L (PBS-0.5 Mini) vertical wheel bioreactors.ResultsA three stage suspension manufacturing process is developed, transitioning from adherent to suspension culture, with TB2 media supporting iPSC growth during scaling. Stage-wise optimization approaches and extended differentiation times are used to enhance marker expression and maturation of iPSC-derived islet-like clusters. Continuous bioreactor runs were used to study nutrient and growth limitations and impact on differentiation. The continuous bioreactors were compared to a Control media change bioreactor showing metabolic shifts and a more β-cell-like differentiation profile. Cryopreserved aggregates harvested from the runs were recovered and showed maintenance of viability and insulin secretion capacity post-recovery, indicating their potential for storage and future transplantation therapies.ConclusionThis study demonstrated that stage time increase and limited media replenishing with lactate accumulation can increase the differentiation capacity of insulin producing cells cultured in a large-scale suspension environment.

Impact of metabolism-related markers on outcomes in ovarian cancer patients: Findings of the MITO16A/MaNGO-OV2 trial

Introduction In ovarian cancer, expression of metabolism-related markers has been investigated in several studies focusing on individual markers; however, a parallel quantitative evaluation of markers mapping to distinct metabolic processes and their prognostic value in large patient cohorts is still lacking. Methods Here, by using immunohistochemistry followed by digital pathology, we investigated the expression of several markers related to glycolysis including monocarboxylate transporter 1 and 4 (MCT1, MCT4), glutamine metabolism (glutaminase, GLS) and hypoxia/acidosis (carbonic anhydrase 9, CA IX) in tissue microarrays of > 300 patients recruited in the MITO16A clinical trial, which involved treatment of ovarian cancer patients with carboplatin/taxol plus bevacizumab. Results Regarding the prognostic impact of these markers, results indicate that GLS expression correlated with progression-free survival, but this effect disappeared when data were corrected for multiple testing. All other markers showed no correlation with clinical outcome. Conclusion These results indicate marked heterogeneity of expression of metabolism-associated markers in ovarian cancer; however, there was a lack of association with clinical benefit after chemotherapy/anti-vascular endothelial growth factor treatment. Notwithstanding the lack of prognostic value, knowledge of the pattern of expression of these biomarkers in tumors can be useful for patient stratification purposes when new drugs targeting these metabolic pathways will be tested.

Succinic Acid Improves the Metabolism of High-Fat Diet-Induced Mice and Promotes White Adipose Browning

Succinic acid plays a crucial role as an essential intermediate in the mitochondrial tricarboxylic acid cycle in mitochondria. In recent years, growing evidence has supported the the important role of succinic acid in fat metabolism. Therefore, we aimed to investigate the effects of succinic acid on adipose tissue metabolism and insulin sensitivity in high-fat diet (HFD)-induced obese mice and try to explore its potential mechanism. We found that the addition of succinic acid (40 mM) to drinking water inhibited the hypertrophy of inguinal white adipose tissue (iWAT) in HFD-induced mice. Furthermore, succinic acid supplementation enhanced insulin sensitivity and improved their glucose tolerance in obese mice. Interestingly, succinic acid supplementation improved lipid metabolism in HFD-fed mice, as shown by decreased serum levels of TG, TC, LDL-C, and increased HDL-C. In addition, succinic acid supplementation increased the expression of browning markers and mitochondria-related genes in iWAT. Further studies showed that the addition of succinic acid to drinking water promotes the browning of iWAT by activating the PI3K-AKT/MAPK signaling pathway. These results suggest that succinic acid has the potential to be used as an effective component for dietary intervention and may, therefore, play an important role in ameliorating and preventing obesity and associated metabolic diseases caused by HFD.

Clinicopathological analysis of sclerosing haemangiomatoid nodular transformation of the spleen: analysis of three cases and a literature review

ObjectiveTo examine the clinicopathological features, immunohistochemical profiles, and differential diagnosis of sclerosing angiomatiod nodular transformation (SANT).MethodsThree cases of SANT of the spleen, diagnosed between 2014 and 2023 at the Affiliated Hospital of Zunyi Medical University, were analysed. Pathological features were assessed using haematoxylin and eosin staining, followed by immunohistochemistry with the EnVision system. Additionally, a review of relevant literature was conducted.ResultsThe study included one male and two female patients aged 40–55 years, with a median age of 47.5 years. All lesions were solitary, with tumour diameters ranging from 4 to 7.4 cm (mean 5.7 cm). Gross examination demonstrated that the masses were well-demarcated from the surrounding splenic tissue, with no evident capsule. The cut surfaces of the masses exhibited irregular, porcelain-white nodules that were tough in consistency, with some areas intermingling with splenic tissue. Microscopic examination revealed round or circular nodules comprising multiple slit-like or sinusoidal capillaries, separated by concentric collagen fibres. The nodules exhibited chronic inflammatory cell infiltration, calcification, haemosiderin deposition, and fibrous connective tissue with hyaline or mucoid changes. Immunohistochemical analysis demonstrated differential expression of markers, including cluster of differentiation (CD) 34, CD31, and CD8, within the sinusoidal nodule areas. Periodic acid-Schiff staining was positive for perinodular collagen deposits, while reticulin staining highlighted nodule profiles and intranodular vessels. None of the patients experienced postoperative recurrence or metastasis, and one patient was on aspirin for thrombocytosis.ConclusionSANT of the spleen is generally considered a rare, benign lesion with angioma-like characteristics. It exhibits exhibiting distinctive histomorphological features within the red pulp. Understanding the differential diagnosis is crucial to prevent missed or incorrect diagnoses.

DLK1 Is Associated with Stemness Phenotype in Medullary Thyroid Carcinoma Cell Lines

Medullary thyroid carcinoma (MTC) is a rare and aggressive tumor, often requiring systemic treatment in advanced or metastatic stages, where drug resistance presents a significant challenge. Given the role of cancer stem cells (CSCs) in cancer recurrence and drug resistance, we aimed to identify CSC subpopulations within two MTC cell lines harboring pathogenic variants in the two most common MEN2-associated codons. We analyzed 15 stemness-associated markers, along with well-established thyroid stem cell markers (CD133, CD44, and ALDH1), a novel candidate (DLK1), and multidrug resistance proteins (MRP1 and MRP3). The ability to efflux the fluorescent dye Hoechst 3342 and form spheroids, representing CSC behavior, was also assessed. MZ-CRC-1 cells (p.M918T) displayed higher expressions of canonical markers, DLK1, and MRP proteins than TT cells (p.C634W). MZ-CRC-1 cells also formed more spheroids and showed less dye accumulation (p < 0.0001). Finally, we observed that DLK1+ cells (those expressing DLK1) in both cell lines exhibited significantly higher levels of stemness markers compared to DLK1− cells (those lacking DLK1 expression). These findings underscore DLK1’s role in enhancing the stemness phenotype, providing valuable insights into MTC progression and resistance and suggesting potential therapeutic implications.

Establishment of Periodontal Ligament Stem Cell-like Cells Derived from Feeder-Free Cultured Induced Pluripotent Stem Cells.

The periodontal ligament (PDL) is a fibrous connective tissue that connects the cementum of the root to the alveolar bone. PDL stem cells (PDLSCs) contained in the PDL can differentiate into cementoblasts, osteoblasts, and PDL fibroblasts, with essential roles in periodontal tissue regeneration. Therefore, PDLSCs are expected to be useful in periodontal tissue regeneration therapy. In a previous study, we differentiated induced pluripotent stem cells (iPSCs) into PDLSC-like cells (iPDLSCs), which expressed PDL-related markers and mesenchymal stem cell (MSC) markers; they also exhibited high proliferation and multipotency. However, the iPSCs used in this differentiation method were cultured on mouse embryonic fibroblasts; thus, they constituted on-feeder iPSCs (OF-iPSCs). Considering the risk of contamination with feeder cell-derived components, iPDLSCs differentiated from OF-iPSCs (ie, OF-iPDLSCs) are unsuitable for clinical applications. In this study, we aimed to obtain PDLSC-like cells from feeder-free iPSCs (FF-iPSCs) using OF-iPDLSC differentiation method. First, we differentiated FF-iPSCs into neural crest cell-like cells (FF-iNCCs) and confirmed that FF-iNCCs expressed NCC markers (eg, Nestin and p75NTR). Then, we cultured FF-iNCCs on human primary PDL cell-derived extracellular matrix for 2 weeks; the resulting cells were named FF-iPDLSCs. FF-iPDLSCs exhibited higher expression of PDL-related and MSC markers compared with OF-iPDLSCs. FF-iPDLSCs also demonstrated proliferation and multipotency in vitro. Finally, we analyzed the ability of FF-iPDLSCs to form periodontal tissue invivo upon subcutaneous transplantation with β-tricalcium phosphate scaffolds into dorsal tissues of immunodeficient mice. Eight weeks after transplantation, FF-iPDLSCs had formed osteocalcin-positive bone/cementum-like tissues and collagen 1-positive PDL-like fibers. These results suggested that we successfully obtained PDLSC-like cells from FF-iPSCs. Our findings will contribute to the development of novel periodontal regeneration therapies.

Utilizing the apical-out enteroids in vitro model to investigate intestinal glucose transport, barrier function, oxidative stress, and inflammatory responses in broiler chickens

IntroductionConventional 2D intestinal epithelial cell lines have been widely used in investigating intestinal functions, yet with limitations in recapitulating the in vivo gut physiology of chickens. A recently established chicken enteroid model with apical-out nature and the presence of leukocyte components represents intestinal mucosal functions. The objectives of this study were to 1) evaluate basic gut nutrient transport and barrier functions in this model and 2) identify the model’s effectiveness in studying inflammation and oxidative stress responses.MethodsEnteroids were generated from individual villus units isolated from the small intestine of Cobb500 broiler embryos. Enteroid viability, morphology, and epithelial cell markers were monitored; barrier function was evaluated based on the permeability to fluorescein isothiocyanate–dextran (FD4) with or without EDTA and lipopolysaccharide (LPS) challenges; nutrient transport was evaluated by fluorescence-labeled glucose (2NBD-G) with or without transporter blockade; the oxidative status was indicated by reactive oxygen species (ROS). Inflammatory and oxidative challenges were induced by LPS and menadione treatment, respectively. Selected marker gene expressions, including tight junction proteins (CLDN-1, CLDN-2, ZO-1, and OCCL), epithelial cell markers (Lgr-5, LYZ, and MUC-2), cytokines (IL-1β, IL-6, IL-8, IL-10, TNF-α, and INF-γ), and antioxidant enzymes (Nrf-2, catalase, and SOD), were determined by using RT-qPCR. Data were analyzed by one-way ANOVA among treatment groups.ResultsEnteroid cell activity was stable from day (d) 2 to d 6 and declined at d 7. Epithelial cell marker and cytokine expressions were stable from d 4 to d 6. FD4 permeability was increased after the EDTA treatment (P ≤ 0.05). Transporter-mediated 2NBD-G absorption was observed, which was reduced with glucose transporter blockade (P ≤ 0.05). Enteroids showed classic responses to LPS challenges, including upregulated gene expressions of IL-1β and IL-6, downregulated gene expressions of ZO-1 and OCCL, and increased FD4 permeability (P ≤ 0.05). Enteroids showed increased ROS generation (P ≤ 0.05) in response to oxidative stress.DiscussionIn conclusion, this apical-out enteroid model is a stable alternative in vitro model that exhibits intestinal barrier, nutrient transport, oxidation, and inflammation functions. With this enteroid model, we developed two challenge protocols for evaluating intestinal functions under oxidative stress and inflammation conditions.

Neuronal fate resulting from indirect neurogenesis in the mouse neocortex

Abstract Excitatory cortical neurons originate from cortical radial glial cells (RGCs). Initially, these neurons were thought to derive directly from RGCs (direct neurogenesis) and be distributed in an inside-out fashion. However, the discovery of indirect neurogenesis, whereby intermediate neuronal progenitors (INPs) generate neurons, challenged this view. To investigate the integration of neurons via these two modes, we developed a method to identify INP progeny and analyze their fate using transgenic mice expressing tamoxifen-inducible Cre recombinase under the neurogenin-2 promoter, alongside thymidine analog incorporation. Their fate was further analyzed using mosaic analysis with double markers in mice. Indirect neurogenesis was prominent during early neurogenesis, generating neuron types that would emerge slightly later than those produced via direct neurogenesis. Despite the timing difference, both neurogenic modes produced fundamentally similar neuron types, as evidenced by marker expression and cortical-depth location. Furthermore, INPs generated pairs of similar phenotype neurons. These findings suggest that indirect neurogenesis, like direct neurogenesis, generates neuron types in a temporally ordered sequence and increases the number of similar neuron types, particularly in deep layers. Thus, both neurogenic modes cooperatively generate a diverse array of neuron types in a similar order, and their progeny populate together to form a coherent cortical structure.

Sonification of Deproteinized Bovine Bone Functionalized with Genistein Enhances Bone Repair in Peri-Implant Bone Defects in Ovariectomized Rats

Estrogen deficiency is one of several contributing factors to catabolic changes in bone surrounding dental implants, impairing bone repair in defects requiring bone regeneration. Functionalizing bone substitutes is an alternative approach among various strategies to address this challenge. In this study, the aim was to evaluate the effect of functionalizing deproteinized bovine bone (Bio-Oss®, BO) with genistein via sonication on peri-implant bone defects in ovariectomized rats. The animals were randomly distributed according to the treatment into the following four groups (n = 10): BO sonicated with genistein (BOS + GEN), BO sonicated alone (BOS), untreated BO (BO), and blood clot only (CLOT). After twenty-eight days, implant removal torque was determined, and the peri-implant bone parameters were calculated based on computed microtomography. Additionally, the gene expression of bone turnover markers was evaluated. As a main result, the functionalization with genistein increased implant removal torque and the peri-implant bone volume in the BOS + GEN group compared to both BOS and BO groups (both p < 0.05). These findings suggest that the sonification of deproteinized bovine bone functionalized with genistein improves bone repair in peri-implant bone defects in ovariectomized rats.

Laboratory analysis of 124 chronic lymphocytic leukemia cases: Single center study

Abstract BACKGROUND: Chronic lymphocytic leukemia (CLL) is a disease characterized by an uncontrolled proliferation of malfunctioning mature B-lymphocytes. It represents a variable range of clinical presentation and prognoses. Understanding the demographic and cellular characteristics of CLL patients can aid in better disease management and treatment outcomes. OBJECTIVES: To identifying hematological parameters and patterns and potential markers that could assist in better diagnosis and of CLL in the local population. PATIENTS, MATERIALS AND METHODS: A retrospective analysis of 124 cases of newly diagnosed CLL was conducted. Data were collected from reports over 2 years (April 2022-April2024), including demographics, Hb levels, WBC counts, platelet counts and expression of CD markers (5, 19, 20, 23, 200, FMC7, LAIR1 and others) using flow cytometry on peripheral blood specimens. Based on FC findings, patients were categorized into two groups for better assessment; definite diagnosis CLL and possible diagnosis CLL; then further subdivided into poor prognosis CLL and good prognosis CLL. RESULTS: Male patients represented 59.5% of cases and females were 40.5% with a median age of 61 years at diagnosis. The mean Hb level was 109 ± 2 g/L, the mean WBC count was 58.0 ± 25.0 × 109/L, and the mean platelet count was 190 ± 40 × 109/L. CD5 was positive in 97.6% of cases, with CD23 in 96%. CONCLUSION: This study provides an overview of the demographic and cellular marker characteristics of CLL patients in FC unit. The findings underscore the heterogeneity of CLL immunophenotype and the importance of detailed characterization in improving patient management protocols; however, further research is warranted to correlate these findings with other parameters of disease burden and prognosis to optimize the therapeutic approaches for CLL patients.

Simultaneous Stimulation of Peripheral Blood Mononuclear Cells with CpG ODN2006 and α-IgM Antibodies Leads to Strong Immune Responses in Monocytes Independent of B Cell Activation

CpG ODN2006 is widely used both in vitro and in vivo to achieve B cell activation and has been previously applied in clinical trials as an adjuvant and anti-cancer agent. Recent studies have demonstrated the benefit of combining CpG ODN2006 with α-IgM antibodies to obtain optimal B cell activation in vitro. In this study, we expanded the knowledge of how both agents affect other types of peripheral blood mononuclear cells (PBMCs), thereby highlighting beneficial and potentially unfavorable properties of the combination of CpG ODN2006 and α-IgM when applied beyond isolated B cells. We elucidated the effects of both compounds on mixed PBMCs, as well as on B cell- and monocyte-depleted PBMCs, allowing us to distinguish between direct effects and indirect influences mediated by other interacting immune cells. Flow cytometry was used to measure the expression of surface markers and intracellular cytokines, while ELISA and multiplex assays were performed to determine cytokine secretion. Our results revealed that stimulation of mixed PBMCs with CpG ODN2006 and α-IgM strongly increased cytokine secretion, primarily originating from α-IgM-stimulated monocytes. Monocyte activation was confirmed by increased CD86 and HLA-DR expression and occurred independently of B cells. The high level of monocyte-derived cytokines after α-IgM exposure did not affect B cell activation. However, it represents a rather unfavorable property for clinical applications. In conclusion, α-IgM is a potent inducer of cytokine production in monocytes. Based on our findings we hypothesize that significant side effects on monocytes can occur when using α-IgM to enhance CpG ODN2006’s efficacy on B cells, particularly in clinical settings.