Antibody Staining Research Articles

P0199 Development of a high-throughput multiplex immunofluorescence and an AI-assisted image analysis tool to study T cells dynamics in pre-clinical rodent models of IBD

Abstract Background T cell therapies are emerging as a new approach to treat and cure patients suffering from immune diseases where T cell homeostasis is no longer maintained. For the evaluation of treatments in pre-clinical rodent models, tools are required that enable fast, observer-independent and high-resolution analysis of different T cells in tissue sections. Multiplex staining of tissue samples provides a valuable opportunity for comprehensive analysis of cellular dynamics with enhanced spatial resolution and tissue context compared to traditional staining and flow cytometry methods. We have developed a high-throughput multiplex immunofluorescence panel for detecting different T cell types and activation status in mouse models of inflammatory bowel disease (IBD). Moreover, we used an AI-assisted method to enhance image analysis, enabling automated, accurate, and quantification of complex cellular interactions. Methods For the development and optimization of the various stainings we used formalin-fixed paraffin embedded (FFPE) mouse colon sections from an efficacy study with murine polyclonal regulatory T cells (Tregs) in the T cell transfer mouse model of IBD, enabling access to sections with healthy versus diseased pathology, as well as samples where Tregs were transferred to reduce colon pathology. Multiplex staining was performed with antibodies (such as CD3, FOXP3, CD25, CD69, CTLA-4 and KI67) using a Leica BOND RX autostainer, by applying sequential primary antibody staining, followed by secondary HRP-conjugated antibodies and Opal-TSA chemistry. Whole slide images (WSI) were generated by a Phenoimager HT (Akoya) and analyzed using Visiopharm software, with DAPI+ nuclei detection via AI-based application. Results We successfully developed a multiplex panel that enables high throughput staining and spatial resolution analysis of over six markers. We were able to determine the activation status of different subtypes of T cells and their specific localization in the colon. We could quantify infiltration of activated effector T cells (Teffs) in both the mucosa and submucosa in colon samples with high pathological scores, as well as FOXP3+ cells in samples from mice treated with Tregs. Conclusion We developed an Opal-based multiplex immunofluorescence method to detect T cells in mouse colon samples, focusing on activation and proliferation markers. Additionally, an AI-assisted image analysis tool that provided fast, reproducible, and quantitative results with spatial resolution. This innovative multiplex method holds promise for adaptation to different mouse models of colitis and other rodent models related to immune diseases, furthering its potential impact in the area of T cell therapy research.

The cortical high-flow sign in oligodendroglioma, IDH-mutant and 1p/19q-codeleted is correlated with histological cortical vascular density.

The cortical high-flow sign has been more commonly reported in oligodendroglioma, IDH-mutant and 1p/19q-codeleted (ODG IDHm-codel) compared to diffuse glioma with IDH-wildtype or astrocytoma, IDH-mutant. Besides tumor types, higher grades of glioma might also contribute to the cortical high flow. Therefore, we investigated whether the histological cortical vascular density or CNS WHO grade was associated with the cortical high-flow sign in patients with ODG IDHm-codel. This retrospective study consisted of pathologically confirmed 25 adult patients with ODG IDHm-codel. We implemented pseudo-continuous arterial spin labeling technique with background suppression. Subtraction images were generated from paired control and label images. Tumor-affecting cortices without intense contrast enhancement on conventional MR imaging were targeted for the determination of the cortical high-flow sign. Immunohistochemical staining of CD31 antibody was performed for the identification of vascular endothelial cells. A microscopic field of the most intense vascularization was captured in each specimen. The vessel number and the relative vascular density (%Vessel) were compared between the positive cortical high-flow sign (CHFS+) and the negative cortical high-flow sign (CHFS-) groups using the Mann-Whitney U test. Second, Fisher's exact test was used to compare the difference between the presence or absence of cortical high-flow sign and CNS WHO grades. Finally, the vessel number and %Vessel were compared between the CNS WHO grade 2 and grade 3 using the Mann-Whitney U test. The vessel number and %Vessel were higher in patients with the CHFS+ group than in patients with CHFS- group (p = 0.016 and p = 0.005, respectively). We observed no significant differences (p = 1.00) in the frequency of cortical high-flow sign between the CNS WHO grade 2 and grade 3. In addition, no significant differences are found in the vessel number and %Vessel between the CNS WHO grade 2 and grade 3 (p = 0.121 and p = 0.475, respectively). The cortical high-flow sign on ASL, which is more commonly found in ODG IDHm-codel than in diffuse glioma with IDH-wildtype or astrocytoma, is associated with the histological cortical vascular density in patients with ODG IDHm-codel.

Mitochondria-Targeted Antioxidant (MitoQ) and Nontargeted Antioxidant (Idebenone) Mitigate Mitochondrial Dysfunction in Corneal Endothelial Cells.

To investigate the effectiveness of mitochondrial-targeted antioxidant mitoquinone (MitoQ) and nontargeted antioxidant idebenone (Idb) in alleviating mitochondrial dysfunction in corneal endothelial cells (CEnCs). In vitro experiments were conducted using immortalized normal human corneal endothelial cells (HCEnC-21T; SVN1-67F) and Fuchs endothelial corneal dystrophy (FECD) cells (SVF5-54F; SVF3-76M). Cells were pretreated with MitoQ or Idb and then exposed to menadione (MN) with simultaneous antioxidant treatment. Mitochondrial parameters were evaluated through adenosine triphosphate viability assays, JC-1 staining for mitochondrial membrane potential, and Tom-20 antibody staining for fragmentation, with analysis performed using ImageJ software. HCEnC-21T cells were additionally exposed to ultraviolet-A (25 J/cm2) to assess drug effects under physiological stress. Mitochondrial fragmentation in FECD specimens was analyzed pre- and post-treatment with the drugs. Statistical analysis was conducted using 1-/2-way analysis of variance with post-hoc Tukey test. MitoQ and Idb enhanced cell viability and mitochondrial membrane potential in both normal and FECD cells under MN-induced stress. Idb reduced MN-induced mitochondrial fragmentation by 32% more than MitoQ in HCEnC-21T cells and by 13% more in SVF5-54F cells. Under ultraviolet-A stress, Idb and MitoQ improved mitochondrial function by 31% and 25%, respectively, with MitoQ increasing mitochondrial function by 42% in FECD specimens. Differential responses in mitochondrial dysfunction across cell lines highlight disease heterogeneity. MitoQ and Idb protected CEnCs from oxidative stress and improved mitochondrial bioenergetics, suggesting that mitochondrial-targeted antioxidants could be considered for mitochondrial dysfunction in CEnCs.

A Comparative Study on Kidney Morphology of Anatolian Ground Squirrels, Rabbits, and Rats

ABSTRACTIn this study, the kidneys of ground squirrels (hibernated and nonhibernated), rabbits, and rats were examined macro and microanatomically. Kidney morphology was investigated by stereo microscopy, light microscopy, and scanning electron microscopy. Triple and immunohistochemical staining were performed for light microscopic examinations. Cold crushing and takilon injection into the renal arteries were performed for scanning electron microscopic examinations. The course and branching of intrarenal capillaries and morphologic features from glomeruli to foramina papillaria were demonstrated. The location, position, shape, weight, and size of the kidneys of the three species studied were determined. Relative medullary thickness (RMT) and kidney index (Ki) values were calculated from the measurements. Based on the RMT values of the three species, it was concluded that they are mammals belonging to the semiarid and humid habitat category. The number of nephrons with long segments was high in the kidneys of all three species. Structural findings suggested that the rat may produce more concentrated urine than the ground squirrel, and the ground squirrel may produce more concentrated urine than the rabbit. The mean diameter of the renal corpuscles was 112.5 μm in the hibernated ground squirrel, 137.6 μm in the nonhibernated ground squirrel, 138.1 μm in the rabbit, and 137.8 μm in the rat. In hibernated ground squirrels, a narrowing of the cavum glomeruli and a decrease in renal corpuscle diameters were found. In contrast to ground squirrel and rat kidneys, rabbit kidneys showed the presence of species‐specific subcapsular glomeruli. Immunohistochemistry (antinestin antibody) staining and scanning electron microscope (SEM) images revealed the structure of podocytes in detail. Species‐specific area cribrosa configurations were detected in the renal papillae of the kidneys we examined. With this study, new renal morphological findings were obtained in ground squirrels, rabbits, and rats.

Morphology and phylogeny of Itaspiella helgolandica, an interstitial marine flatworm with circumpolar distribution

Flatworms in the family Otoplanidae Hallez, 1892, are ubiquitous inhabitants of the swash zone of marine sandy beaches worldwide. We present new insights into the morphology and phylogenetic position of the otoplanid Itaspiella helgolandica (Meixner, 1938) based on specimens sampled in northern Norway. While the species was originally described from the German coast, it has since been reported in the High Arctic as well as in Greenland, Canada and Alaska, indicating that it has a circumpolar distribution. Apart from these reports, I. helgolandica has received marginal scientific attention beyond the original species description, and apart from drawings, there is not much detailed data available of its morphology. We employ antibody staining and confocal laser microscopy to provide detailed morphological information on the ciliation, myoanatomy and genital organs of specimens of I. helgolandica and compare their morphology with other accounts of the species from across the Arctic. We investigate the phylogenetic relationships of Itaspiella Ax, 1955 through a Bayesian/RAxML analysis of 28S sequences and confirm the morphologically underpinned notion that this genus sits within the subfamily Otoplaninae Hallez, 1910, as the sister taxon to Xenotoplana Ax, Weidemann & Ehlers, 1978. In light of our findings, we explore the question of the apparent circumpolar distribution of Itaspiella in the context of the larger “meiofauna paradox” and touch upon various evolutionary phenomena that may have contributed to phenotypic stability despite its limited dispersal capabilities and the vicariance imposed by the complex recent geological history of the Arctic.

The tumour histopathology "glossary" for AI developers.

The applications of artificial intelligence (AI) and deep learning (DL) are leading to significant advances in cancer research, particularly in analysing histopathology images for prognostic and treatment-predictive insights. However, effective translation of these computational methods requires computational researchers to have at least a basic understanding of histopathology. In this work, we aim to bridge that gap by introducing essential histopathology concepts to support AI developers in their research. We cover the defining features of key cell types, including epithelial, stromal, and immune cells. The concepts of malignancy, precursor lesions, and the tumour microenvironment (TME) are discussed and illustrated. To enhance understanding, we also introduce foundational histopathology techniques, such as conventional staining with hematoxylin and eosin (HE), antibody staining by immunohistochemistry, and including the new multiplexed antibody staining methods. By providing this essential knowledge to the computational community, we aim to accelerate the development of AI algorithms for cancer research.

Are Ki-67 and Procalcitonin Expression Levels Useful in Predicting the Biological Behavior of Hepatocellular Carcinoma After Liver Transplantation?

Background: Examinations of procalcitonin (PCT) and Ki-67 expression levels in hepatocellular carcinoma (HCC) patients who have undergone liver transplantation (LT) through immunohistochemical analyses of tumor tissue may reveal the biological characteristics of the tumor, thus informing the selection of HCC patients for LT. Methods: Hepatectomy specimens from 86 HCC patients who underwent LT were obtained and analyzed immunohistochemically for the expression of PCT and Ki-67. The percentage and intensity of PCT staining, as well as the percentage of Ki-67 expression, were assessed for each patient. The impacts of PCT and Ki-67 expression on disease-free survival, overall survival, and the recurrence rate were studied, as well as their correlations with other clinicopathological features. Results: The recurrent HCC group showed a higher Ki-67 level (p < 0.001), larger maximum dominant tumor diameter (p < 0.001), and higher rate of vascular invasion (p = 0.001). The pre-transplant AFP (p = 0.001), maximum dominant tumor diameter (p < 0.001), number of tumor nodules (p < 0.001), rate of vascular invasion (p = 0.001), and Ki-67 level (p = 0.044) were higher in patients beyond the Milan criteria. Similarly, the pre-transplant AFP (p < 0.001); maximum dominant tumor diameter (p < 0.001); number of tumor nodules (p < 0.001); rates of portal vein tumor thrombus (p = 0.002), poor differentiation (p = 0.021), and vascular invasion (p < 0.001); and Ki-67 level (p = 0.010) were higher in patients beyond the expanded Malatya criteria. The maximum dominant tumor diameter (p = 0.006); Ki-67 level (p = 0.003); rates of vascular invasion (p < 0.001), cases beyond the Milan criteria (p = 0.042) and the expanded Malatya criteria (p = 0.027), and portal vein tumor thrombus (p = 0.020); and presence of recurrence (p < 0.001) were higher in HCC patients with mortality. The Kaplan-Meier estimates indicated that Ki-67 levels exceeding 5% significantly affected DFS and OS. Although the Kaplan-Meier estimates indicated that a PCT staining percentage of ≥25% did not have a statistically significant effect on DFS or OS, the outcomes may be considered clinically significant. Conclusions: This study demonstrated that the Ki-67 proliferation index can be used as a predictive biomarker of the biological behavior of HCC. Furthermore, we claim that PCT expression over a particular threshold might impact recurrence and survival, and we believe that further multicenter prospective studies focused on standardized PCT antibody staining are crucial in order to determine its potential as a biomarker for HCC.

A Pair of Fluorescent Probes Enabling Precise Diagnosis of Liver Cancer by Complementary Imaging.

Hepatocellular carcinoma (HCC) is by far the predominant malignant liver cancer, with both high morbidity and mortality. Early diagnosis and surgical resections are imperative for improving the survival of HCC patients. However, limited by clinical diagnosis methods, it is difficult to accurately distinguish tumor tissue and its boundaries in the early stages of cancer. Herein, we report two fluorescent probes, cLG and hLR, for the detection of cancer and healthy cells, respectively, enabling the precise diagnosis of liver cancer by providing complementary imaging. These two fluorescent probes could selectively stain the target cells in the liver tissue imaging, which is confirmed by H&E and antibody staining. Moreover, for the first time, the cancerous area and healthy area are clearly identified by the cocktail of these two probes, suggesting its potential to be used in fluorescence-guided surgery. Finally, we identify transporter SLC27A2 as the gating target of cLG through a systematic transporter screen using a CRISPR activation library. SMPD1 was identified as the target of hLR through a thermal proteome profiling. Therefore, the development of these two highly specific probes offers complementary imaging and provides a unique diagnostic tool for cancer disease, even for fluorescence-guided surgery.

Evaluation of the antibacterial activity of some plant essential oils against Ralstonia solanacearum and the molecular diagnosis of the bacterium

Background Evaluation of the antibacterial activity of some essential oils for controlling potato bacterial wilt. Objective In vitro and planta trials, five essential oils, lemongrass, eucalyptus, thyme, ginger, and peppermint, were evaluated for their aptitude to impede the progress of Ralstonia solanacearum. Materials and methods Five essential oils were assessed for their activity to inhibit R. solanacearum growth using the disc diffusion method. Certain volumes of each tested plant essential oil were added on filter paper discs using tetrazolium chloride agar medium, which contained the tested bacterium to obtain the proposed concentrations used to evaluate the most efficient oil for controlling potato bacterial wilt. Serological and molecular identification was also conducted. Results and conclusion R. solanacearum was characterized using cultural characteristics on the selective medium South Africa, “which is the most effective in suppressing the growth of contaminating microorganisms, and the highest recovery rate of the tested bacterium,” immunofluorescence antibody staining tomato bioassay test and molecular identification via real-time PCR (Taq-man) assay. In-vitro studies showed that all tested plant essential oils (lemongrass, eucalyptus, thyme, ginger, and peppermint) significantly reduced R. solanacearum progress. Eucalyptus (Eucalyptus globulus) and peppermint (Mentha piperita) essential oils were the most efficient in suppressing R. solanacearum growth, where it showed the highest mean inhibition zone, followed by thyme oil (Thymus vulgaris), lemongrass oil (Comybogen citratus), and ginger oil (Zingiber officinale), respectively. Phenotypic variances in bacterial cell construction have been noticed via a transmission electron microscope. Eucalyptus oil led to cell wall lysis, cell laceration, bubble-like structures, and degraded cellular components in bacterial cells.

Bcl-2 and galectin-3 expression is associated with recurrence of ameloblastoma.

Ameloblastoma is a benign odontogenic neoplasm with a high recurrence rate. Identifying cellular and molecular changes in this neoplasm may help predict the recurrence risk. Bcl-2 and galectin-3 are anti-apoptotic proteins associated with the prognosis of many neoplasms. However, there are a few studies focusing on the association between these two markers and recurrence of ameloblastoma. This study aimed to investigate the association of Bcl-2 plus galectin-3 expression and recurrence of ameloblastoma. This retrospective cross-sectional study was designed on 48 paraffin-embedded blocks diagnosed as ameloblastoma from 1998 to 2019. We retrieved follow-up data from patients' records and used immunohistochemical staining for Bcl-2 and galectin-3 antibodies. Then, we analyzed their association with recurrence using Chi-square and Mann-Whitney test as well as recurrence-free survival using Kaplan-Meier curves and linear Cox regression. The level of statistical significance was P < 0.05. Twenty-six patients had experienced the recurrence. The mean follow-up time was 93.53 months. There was a significant association between Bcl-2 plus cytoplasmic galectin-3 staining and recurrence (both P < 0.001). Furthermore, in univariate analysis, high expression of Bcl-2 was associated with less recurrence-free survival (log-rank: P = 0.020-univariable Cox: P = 0.033), but in multiple Cox regression, there was no significant association (P = 0.471). High cytoplasmic galectin-3 expression was also associated with less recurrence-free survival (log-rank: P = 0.007-univariable Cox: P = 0.015-multiple Cox: P = 0.044). Furthermore, we found a correlation between Bcl-2 and cytoplasmic galectin-3 staining (P = 0.001). It seems that Bcl-2 and cytoplasmic galectin-3 staining might predict the risk of ameloblastoma recurrence. However, only the cytoplasmic galectin-3 staining might be an independent predictor of ameloblastoma recurrence, and we recommend further studies.

Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1.

Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca2+ mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca2+ mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.2 % was set. The resting baseline relative fluorescent unit (RFUs) signals of the TRPM8 Ca2+ mobilization assay exhibited substantial well-to-well variability, even though such differences were small relative to maximal agonist induced responses. Maximum RFU, cumulative RFU sum, or area under the curve values were extracted from Ca2+ mobilization kinetic data to plot curves and calculate EC50 and IC50 values. Fold over baseline (FOB) ratio data processing eliminated well-to-well differences in resting baseline signals, reduced error bars, improved curve fits and reduced 95 % confidence interval EC50 and IC50 ranges. FOB ratio data processing decreased variability and improved the precision of repeat measurements in single experimental sessions thereby reducing the minimum threshold difference in EC50 or IC50 values required to distinguish compound potencies. EC50 and IC50 values of TRPM8 agonists and antagonists determined in single experiments were strongly aligned to those from multiple independent experiments. Benchmark TRPM8, TRPV1, and TRPA1 EC50 and IC50 values were within the ranges previously reported for agonist and antagonist standards. The improved precision and accuracy of the TRP Ca2+ mobilization assays afforded by FOB ratio data processing enhances their utility for investigating structure activity relationships.

O-GlcNAcylation promotes astroglial-mesenchymal transition via the connexin43 pathway under high-glucose conditions.

This study aimed to investigate the effects of O-linked N-acetylglucosamine modification (O-GlcNAcylation) on astroglial-mesenchymal transition through connexin43 (Cx43) pathway under high-glucose conditions. The primary rat astrocytes were cultured under normal and high-glucose conditions, and level of GFAP, α-SMA and Cx43 was investigated. To explore the influence of O-GlcNAcylation on astroglial-mesenchymal transition, Thiamet G treatment was employed to enhance O-GlcNAcylation, while Alloxan was used to decrease it. Cx43 knockdown was acquired through lentivirus constructs to explore its role in astrocyte transition. The levels of GFAP and α-SMA expressions were examined, while astrocyte proliferation was evaluated using the CCK-8 assay, and migration was assessed through wound healing assays. The results showed that primary rat astrocytes were identified by GFAP antibody staining. Under high-glucose conditions, the levels of GFAP, α-SMA, and Cx43 increased, as confirmed by Western blot and immunofluorescence. O-GlcNAcylation augmentation induced by Thiamet G treatment significantly increased the expression of GFAP, α-SMA, and Cx43 compared to both normal and high-glucose conditions. Conversely, the inhibition of O-GlcNAcylation reversed the high-glucose-induced increase in GFAP and α-SMA. Cx43 knockout led to the downregulation of GFAP and α-SMA compared to high-glucose and O-GlcNAcylation-augmented conditions. Additionally, levels of O-GlcNAcylation and VEGF were reduced in Cx43 knockout group. Consistently, CCK8 and wound healing assays demonstrated that Cx43 knockout could inhibit astrocyte proliferation and migration compared to the high-glucose and O-GlcNAcylation augmented groups. These findings demonstrate that astroglial-mesenchymal transition occurs under high-glucose conditions, and can be promoted by O-GlcNAcylation augmentation, but suppressed by Cx43 knockout. The study underscores the significant role of Cx43 in this transition and its potential involvement in diabetic complications.

Examination of antinuclear antibody staining patterns and titers in patients with childhood-onset systemic lupus erythematosus.

Antinuclear antibodies (ANA) staining patterns can provide useful information in systemic lupus erythematosus (SLE). In our study, we examined the frequency of ANA staining patterns in disease-related features in childhood-onset SLE patients. ANA and its staining patterns were assessed in childhood-onset SLE patients. Two hundred twenty-three patients were included (F/M = 3/1). Their median age at diagnosis was 14.3 (11.9-16.1) years. The anti-cell (AC)-4/5 (fine or large speckled) pattern was the most common nuclear ANA pattern (75.8%), while the AC-19 (dense fine speckled) pattern was the most frequently detected cytoplasmic ANA pattern (13.1%). The AC-4/5 (fine or large speckled) patterns were notably seen in fever, acute and chronic cutaneous lupus, arthritis, serositis, hematologic involvement, renal involvement, neuropsychiatric involvement, gastrointestinal involvement, and cardiopulmonary involvement (all p < .001). Conversely, the AC-1 (homogeneous) pattern was significantly detected in oral/nasal ulcers and non-scarring alopecia (both p < .001). Regarding the laboratory features, the AC-4/5 (fine or large speckled) patterns exhibited a predominant seen in autoimmune hemolytic anemia, leukopenia, thrombocytopenia, elevated ESR and CRP, hypocomplementemia, direct Coombs, anti-Smith (Sm), anti-SSA and SS-B, anti-ribonucleoprotein (RNP), anti-histone, anti-ribosomal P, lupus anticoagulant, anti-cardiolipin immunoglobulin (Ig)M/IgG, and anti-β2-glycoprotein IgM/IgG positivities (all p < .001). In contrast, the AC-1 (homogeneous) pattern was detected in anti-double-stranded (ds) DNA and anti-histone positivity (both p < .001). Our study showed that AC-4/5 and AC-1 patterns of ANA are frequently detected in many clinical and serological features of childhood-onset SLE patients. However, further studies are needed in larger populations to verify these results.

Influence of Bovine Myosin Heavy Chain Isoforms and Muscle Fiber Cross-Sectional Are&lt;em&gt;a o&lt;/em&gt;n the Eating Quality and Connective Tissue Characteristics of 11 Different Beef Muscles

The objective of this study was to determine the impact of muscle fiber type, cross-sectional area (CSA), and diameter on the eating quality of 11 different beef muscles. Eleven different beef muscles were utilized in 2 separate studies. In the 2 studies, triceps brachii, rectus abdominus, rectus femoris, supraspinatus, gluteus medias, pectoralis profundi, semitendinosus, longissimus thoracis, longissimus lumborum, tensor fascia latae, and gastrocnemius were collected from 10 USDA Choice carcasses (N = 110). To determine muscle fiber type, myofibrillar proteins were extracted and separated via gel electrophoresis and immunoblot, while muscle fiber CSA and diameter were determined using a dystrophin antibody stain via fluorescence microscopy. Pearson correlation analysis was performed to determine the relationship between muscle fiber type, CSA, diameter, and the eating quality of the 11 beef cuts from previously reported studies. Muscles from both studies showed distinct differences in the relative percentage of type I and type IIA muscle fiber types, CSA, and diameter (P &amp;lt; 0.05). Correlation analysis from study 1 demonstrated positive correlations between type I fibers and many positive attributes of eating quality such as tenderness, juiciness, and lipid flavor intensity, while negative correlations were found between type IIA fibers and those attributes (P &amp;lt; 0.01). Interestingly, results from study 2 showed that increasing type I fiber percentage may also contribute to greater connective tissue content and collagen crosslink density (P &amp;lt; 0.01). Finally, a negative correlation was found between muscle fiber CSA and diameter with connective tissue amount (P &amp;lt; 0.05), and a positive correlation was found between muscle fiber CSA and diameter with tenderness measurements (P &amp;lt; 0.05) in both studies. Overall, muscles with greater type I fiber % delivered a more favorable eating experience than those with more glycolytic metabolism. Notably, increased CSA and fiber diameter did not diminish eating quality and were found to have a muscle-specific relationship with tenderness.

Pre-vascularisation of the acellular bladder matrix using the omentum in a porcine model prior for bladder reconstruction-a step towards clinical application?

Research studies with porcine acellular bladder matrix (PABM) showed integration of only small sized stamps in recipient bladders, however for clinical use in bladder augmentation significantly larger patches are needed. We hypothesised pre-vascularisation with omentum may be a step towards clinical translation. Eight domestic pigs were operated three times 8-10weeks apart: 1-Implantation; PABM with recorded dimensions were sutured around a tissue expanding device, wrapped in omentum and sutured to the anterior abdominal wall. 2-Augmentation; hemi-cystectomy and bladder augmentation was performed with the pre-vascularized PABM using non-absorbable suture 3-Sacrifice; The dimensions of the PABMs were measured macroscopically, the in-vivo microcirculation of the PABMs were assessed using laser speckle contrast imaging. HE staining, uroplakin 3 and CK7 immunohistochemistry was performed. In seven animals, the bladder augmentation was successful without complication. One animal was lost in bowel obstruction and in two animals enteric fistula was found after the first intervention. The rectangular shape of the initial tissue expander was subsequently changed. All the seven patches were strong, compliant and had integrated with the surrounding native bladder and were 83% (48-100%) of the original patch size. Laser speckle contrast imaging already showed microcirculation at the patch edges at augmentation and this further improved towards the centre of the patches by the end of the study. Histology demonstrated acute inflammatory response with fibroblast infiltration after implantation and full coverage by urothelium was seen with positive staining for CK7 antibodies. Pre-vascularization of PABM in the omentum of healthy porcine models allows larger PABM patches to integrate this may be a step towards clinical application.

Synthesis of SulfoCy5‐ and SulfoCy7‐αGalCer Probes as Chemical Tools for Investigating the Uptake of Liposomal αGalCer Conjugates by Antigen Presenting Cells

Abstractα‐Galactosylceramide (αGalCer) is a synthetic glycolipid that, when loaded into the CD1d receptor of antigen presenting cells, activates iNKT cells to coordinate the generation of both innate and adaptive immune responses. In vaccines, the αGalCer adjuvant is generally delivered in liposomes, but interestingly, little is known about the intracellular fate of αGalCer‐antigen conjugates resulting in the absence of clear “design rules” for this type of compounds and their formulations. To unravel the uptake mechanism of liposomal αGalCer conjugates and understand the fate of its components, we synthesized sulfoCy7‐αGalCer 1 and sulfoCy5‐αGalCer 2. The fluorescent probes were obtained from a linker‐functionalized αGalCer, which was then modified with sulfoCy7/sulfoCy5. After formulation in liposomes, the cellular presentation was studied in a murine dendritic cell line using fluorescence imaging and flow cytometry. Imaging indicates that liposomes loaded with sulfoCy5‐αGalCer are taken up by DC2.4 cells in a heterogeneous and concentration‐dependent manner. Additionally, fluorescence antibody staining confirms that αGalCer is ultimately presented by CD1d receptors, but importantly is cleaved from sulfoCy5 before reaching the cell surface. These results validate our synthetic probes as useful in mechanistic studies of the uptake of αGalCer‐antigen conjugates as shown here for DC2.4 cells.

Abstract 344: Novel technique for endovascular biopsy of carotid endothelial cells in carotid artery stenosis: Proof of Concept &amp; Follow Up of SVIN 2023 Pilot Grant

Introduction Acute ischemic stroke remains to be one of the top causes for morbidity and mortality among the general population, with approximately 800,000 strokes occurring per year nationwide. While the potential etiologies of stroke are numerous, 15‐20% of all strokes are attributed to carotid artery stenosis. While isolated studies have been conducted sampling carotid plaques after CEA, there is a dearth of literature examining the characteristics of the endoluminal cells and their molecular characteristics (such as transcriptomes) in carotid artery disease. We therefore employed a novel method for endovascular biopsy of carotid endothelial cells in the setting of carotid artery stenosis. Methods A prospective registry of adult patients undergoing endovascular treatment for carotid artery stenosis was maintained from January 2024 to August 2024. Informed consent was obtained, and at the conclusion of the case, critical devices were collected and placed in dissociation buffer including the access wire/sheath tip, the distal embolic protection device, and any balloons utilized for balloon angioplasty. Cells were then washed from the samples using a series of centrifugation, wash, and pelleting steps. Once completed, the pellet was then resuspended in a solution containing Hibernate A, and a standard antibody staining and sorting protocol was performed utilizing fluorescence activated cell sorting (FACS) to identify endothelial cells (via CD31 antibodies) and to exclude white blood cells (negative sorting via CD45 antibodies). Results Six patients met inclusion criteria and gave consent for endothelial carotid biopsy. After sorting for endothelial cells via FACS, the mean number of endothelial cells collected from the access sheath/wire was 1506 cells per patient. Similarly, the mean number of endothelial cells collected from the balloons utilized for angioplasty was 1124 cells. Finally, the mean number of endothelial cells collected from the distal embolic protection device was 5021 cells per patient. Discussion The preliminary findings herein suggest that this method of endovascular biopsy may very well be a reliable method for collecting endothelial cells from the carotid artery in the setting of treatment for carotid artery stenosis. Sample collection is still ongoing to further support the viability of this technique. Collected endothelial cells will then undergo RNA sequencing in order to describe the transcriptomic profile present in endothelial cells of diseased carotid arteries.

Multiplexed Immunophenotyping of Lymphoma Tissue Samples.

High-plex imaging techniques enable the detection and quantification of a multitude of markers in tissue biopsies at single-cell or near-single-cell resolution. In lymphoma, this can facilitate the detection and characterization of cellular phenotypes and interactions, describing both tumor and microenvironmental cells. In combination with other techniques, high-plex imaging allows the investigation of biological mechanisms and clinically relevant biomarkers. CO-Detection by IndEXing (CODEX), one of such techniques, is based on antibodies labeled with unique DNA oligonucleotides that can be visualized by complementary reporter oligonucleotides coupled to a fluorophore. Here, we provide an overview of the key steps of a CODEX-based project, including (1) antibody panel design, (2) cohort selection, (3) staining and imaging, (4) data analysis. By sharing our CODEX protocol and our experience with FFPE tissue samples, we aim to encourage wider use of this powerful technique in lymphoma research and improve insight into cellular composition and spatial dynamics for improved diagnostics and therapy.

8637 Pituicytoma mimicking Craniopharyngioma

Abstract Disclosure: O. Araoye: None. T. Delibasi: None. A. Chaugule: None. S.K. Dhillon: None. C. Fuller: None. Pituicytoma, a rare low-grade glial tumor originating from the neurohypophysis or infundibulum of the pituitary gland, poses diagnostic challenges due to its propensity to mimic other sellar or suprasellar lesions. We present a case of a 51-year-old male with a history of a suprasellar mass initially suggested to be a craniopharyngioma, incidentally discovered. Opting for elective surgery, the patient underwent transsphenoidal resection, revealing a highly vascular tumor adherent to the optic chiasm and pituitary stalk. While the tumor was successfully removed, sacrifice of the pituitary stalk led to postoperative complications, including cerebrospinal fluid (CSF) leak and central diabetes insipidus (DI).Histopathological examination characterized the tumor as oncocytic pituicytoma/spindle cell oncocytoma, a subtype of pituicytoma. The anti-mitochondrial antibody staining at an external laboratory displayed diffuse granular cytoplasmic staining supporting oncocystic pituicytoma/spindle cell oncocytoma.Postoperatively, the patient developed central DI treated initially with desmopressin (DDAVP), complicated by subsequent hyponatremia necessitating salt tablets and fluid restriction. Additionally, central adrenal insufficiency was managed with hydrocortisone, and central hypothyroidism with levothyroxine. A history of testosterone deficiency was addressed through testosterone injections every 10 weeks.Pituicytoma's diagnostic complexity arises from nonspecific radiological features, requiring reliance on histology and immunohistochemistry for definitive diagnosis. The expression of TTF1 emerges as a characteristic marker. Surgical resection, the primary treatment modality, proves curative in most cases, albeit with associated morbidity including CSF leak, DI, and panhypopituitarism. The role of adjuvant radiotherapy remains uncertain, potentially considered in cases of incomplete resection or recurrence.This case emphasizes three key aspects: first, the potential misdiagnosis of a lobulated suprasellar mass as craniopharyngioma when it could be pituicytoma; second, the development of postoperative DI due to pituitary stalk involvement; and third, the importance of considering pituicytoma in the differential diagnosis of sellar and suprasellar masses.In conclusion, pituicytoma, though rare, demands a nuanced diagnostic approach and comprehensive postoperative management to address potential complications and optimize patient outcomes. Presentation: 6/2/2024

Imaging Mass Cytometry in Psoriatic Disease Reveals Immune Profile Heterogeneity in Skin and Synovial Tissue

Imaging Mass Cytometry (IMC) is a technology that enables comprehensive analysis of cellular phenotypes at the tissue level. We performed a multi-parameter characterization of structural and immune cell populations in psoriatic skin and synovial tissue samples aimed at characterizing immune cell differences in patients with psoriasis, psoriatic arthritis (PsA).A panel of 33 antibodies was used to stain selected immune and structural cell populations. IMC data were segmented into single cells based on combinations of antibody stains. Single cells were then clustered into cell categories based on pre-specified markers. The spatial relationships of different cell populations were assessed using neighborhood analysis.Among all cell types in the skin and synovium, lymphoid cells accounted for the most prevalent cell type. T cells and macrophages were the most prevalent immune cell type in the synovium and B cells and NK cells were also identified. Neighborhood analysis showed high correlation between synovial T cells, B cells, macrophages, dendritic cells and neutrophils suggesting spatial organization.Innate and adaptive immune cells can be reliably identified using IMC in skin and synovium. Inter-patient heterogeneity exists in tissue cell populations. IMC provides opportunities for exploring in depth underlying immunological mechanisms driving psoriasis and PsA.