Differences In Staining Intensity Research Articles

Evaluation of ABCB5 immunostained epithelial stem cells in oral squamous cell carcinoma, inflammatory gingival hyperplasia and normal mucosa

ABSTRACTOral cancer is the most prevalent head and neck cancer. Although tumor markers have been investigated for detecting the progression and prognosis of oral cancer, no reliable marker has been identified. We investigated the expression of ATP binding cassette subfamily B member 5 (ABCB5) positive stem cells in oral squamous cell carcinoma (OSCC) and in inflammatory gingival hyperplasia. We used tissue samples from normal subjects, patients with inflammatory gingival hyperplasia, and patients with OSCC. Samples were investigated using anti-ABCB5 monoclonal antibody immunohistochemistry to detect epithelial stem cells. Staining density, intensity, and immunoreactive scores of ABCB5 were analyzed for the three study groups. We found ABCB5 immunostaining in all three study groups, but different distributions of ABCB5 expression in different layers of the epithelium. We found no significant difference in staining intensity between inflammatory hyperplasia and normal mucosa, but we found significantly stronger expression in OSCC compared to normal and inflammatory hyperplasia individually. Elevated expression of ABCB5 in OSCC suggests an increased subpopulation of tumor cells with an undifferentiated stem cell phenotype, which facilitates cancer initiation and progression.

Comparison of the IHC Markers CD138 and CD43 in Oral Leukoplakia: An Original Research.

In the clinical practice, one of the most common suspicious lesions that may be potentially malignant is oral leukoplakia. Globally, the rate at which it turns malignant varies. This study examines the levels of markers CD138 and 43 in oral leukoplakia. Twenty archival blocks of confirmed epithelial dysplasia were taken from the Department of Oral Pathology. These were processed for the identification of markers CD138 and 43 through Immuno Histo Chemistry (IHC). The blocks were divided equally for both the markers. There was a noticeable difference in staining intensity between dysplastic tissue and nondysplastic epithelium. However, CD138 expression was low or weak in dysplastic epithelium. CD43 expression was negative in all nonhematopoietic tissues. Genes that are cancer associated have been found to have incredibly different impacts in numerous tissues during the multistep process of oral carcinogenesis. In tissues undergoing dysplastic changes, CD138 expression was shown to be decreased, which could point out the malignant changes initiated in the epithelium of the oral tissues.

Immunohistochemical Evaluation of Candidate Biomarkers for Fluorescence-Guided Surgery of Myxofibrosarcoma Using an Objective Scoring Method.

Myxofibrosarcoma (MFS) is the most common soft-tissue sarcoma subtype in elderly patients. Local recurrence (LR) remains a major concern as the lack of intraoperative guidance and an infiltrative growth pattern with long, slender tails hamper surgeons' ability to achieve adequate resection margins for MFS. Fluorescence-guided surgery (FGS) could overcome this concern by delineating tumor tissue during surgery. One of the most important steps to successful FGS is to define a tumor-specific biomarker that is highly overexpressed in tumor tissue while low or absent in adjacent healthy tissue. The aim of this study is to evaluate the expression of eight previously selected promising biomarkers for FGS in MFS tissue samples with adjacent healthy tissue using immunohistochemistry (IHC). The following eight biomarkers were stained in seventeen paraffin-embedded MFS samples: tumor endothelial marker-1 (TEM-1, also known as endosialin/CD248), vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1), vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Flk1), vascular endothelial growth factor-A (VEGF-A), epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), platelet derived growth factor receptor-α (PDGFR-α), and cluster of differentiation 40 (CD40, also known as TNFRSF5). A pathologist specializing in sarcoma annotated the margin between the tumor and adjacent healthy tissue in each MFS tissue sample. Subsequently, we used an objective IHC scoring method to assess and compare the difference in staining intensity between the tumor and adjacent healthy tissue, which is crucial for the use of FGS. TEM-1, VEGF-A, and PDGFR-α stained all MFS tumors, while the other biomarkers did not show expression in all MFS tumors. Ultimately, TEM-1 was identified as the most suitable biomarker for FGS in MFS based on higher tumor-to-background (TBR) staining intensity compared to VEGF-A and PDGFR-α, regardless of preoperative therapy. TEM-1-targeted FGS tracers should be further investigated to optimize MFS treatment.

Difficulties and Recommendations for AI-Based Prediction of Prostate Cancer Aggressiveness in Digital Pathology

Prostate cancer is among the most common cancers in men with around 1.4 million new cases each year world-wide. A vital part in the diagnosis of prostate cancer is the evaluation of its severity using biopsies and histopathology. Recent progress in artificial intelligence-based image analysis has led to a flurry of algorithms for the automated analysis of prostate cancer histopathological data focusing on the detection of cancerous areas, the grading of cancer severity, and patient outcome. Some of these approaches have reached human expert-level performance and digital models trained directly on patient outcomes might surpass human performance in the future. Although these results hold great promise for the future usage of digital pathology in clinical settings, several bottlenecks remain to be addressed. Especially the robustness, reliability and trustworthiness of predictions must be guaranteed across a wide range of variation in protocols and instrumentation. While human experts are relatively robust to technical and biological variation in biopsies, artificial intelligence-based systems tend to struggle with differences in staining intensity, color, scanner type, and image resolution, impeding the clinical usage of digital models. In this work we highlight salient problems and minimal requirements of computational pathology for future use in clinical settings, while focusing on prostate cancer as a use case. In particular, we highlight data and model problems and solutions that include data variability, dataset size, and data annotations, as well as model robustness to data heterogeneity, model prediction confidence, and the explainability of model decisions. While model and data requirements for successful computational pathology in clinics will be highlighted, legal, ethical, and deployment requirements will not be addressed in this review. In summary, we provide a short overview of the field, salient problems, and potential solutions to harvest the full potential of digital pathology for prostate cancer in clinical practice.

Comparative evaluation of host immune response and cytokine signature pertaining to Th1 and Th2 immune arms in serum and tissue among patients of acute localised vs. chronic disseminated dermatophytosis.

India is witnessing an epidemic of dermatophytosis. The role of host immune response against fungi in chronicity and dissemination is topic of ongoing research. We conducted cross-sectional comparative study to determine the difference in Th1 (IFN-γ) and Th2 (IL4) response in serum and tissue between acute and localised vs. chronic and disseminated cases. Patients (18-60 years) were divided in two groups-group A (n=114, BSA <5%, single anatomic site, duration <6 months, n=118) and group B (n=107 BSA >10%, > one anatomic site, duration >12months, n=118). Clinical parameters along with serum levels of IgE, IL-4 and IFN-γ and expression of IL4 and IFN-γ in dermal infiltrate were compared between group. Trichophyton mentagrophytes complex was commonest causative fungi. Serum levels of IgE were significantly higher (median A-539.2, B-2901.0, p < .001) whereas levels IL-4(median A-21.3, B-20.4, p < .001) and IFN-γ(median A-9.6, B-5.1, p < .001) were significantly lower in chronic cases. Expression of IL-4 was observed in most biopsy specimens in both groups without any difference in intensity of staining. Expression of IFN-γ was not detected in all but one specimen across both the groups. Severe itching (OR:0.050, CI:0.018, 0.139, p < .001), sign of topical steroid abuse (OR:0.203,CI:0.077, 0.537; p=.001), ↓IFN-γ (OR:4.683, CI:1.634, 13.418; p=.004) correlated significantly and independently with chronic dermatophytosis. Our study shows chronic and disseminated cases of dermatophytosis differ immunologically in terms of higher IgE, and lower IL4 and IFN-γ. Expression of IL4 is present in tissue of both acute, localised and chronic disseminated cases. Expression of IFN-γ cannot be established in our study.

Clinicopathologic and molecular features associated with response to anti-HER2 therapy in endometrial cancer.

5584 Background: Endometrial cancer (EC) is the most common gynecologic malignancy in the United States. Human epidermal growth factor 2 (HER2) overexpression or gene amplification occurs in 10-60% of EC. The addition of trastuzumab to carboplatin-paclitaxel chemotherapy improves survival in women with HER2-positive serous EC. However, criteria for assessment of HER2 status in EC are not standardized, and furthermore, intratumor heterogeneity may impact therapeutic efficacy. We aimed to identify factors associated with treatment response and survival outcomes in a retrospective cohort of ECs treated with anti-HER2 therapy. Methods: Patients with advanced/recurrent EC treated with trastuzumab or trastuzumab emtansine alone or in combination with chemotherapy from 2013-2021 at our institution were identified. Clinical and treatment information were retrieved from medical records. Central pathology review, HER2 immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) were performed. HER2 IHC was scored using modified ASCO/CAP 2007 guidelines for breast cancer. HER2 expression heterogeneity was defined as a 2-degree difference in staining intensity in ≥5% of tumor cells or discordant IHC score on different specimens. HER2 amplification (tumor/normal fold change) was also estimated from targeted next-generation sequencing (NGS). Best response was assessed by RECIST v1.1 as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD), with clinical benefit defined as CR/PR/SD. Results: Among 39 patients included, median age was 69. Histologic subtypes included serous (n = 18, 46%), carcinosarcoma (n = 7, 18%), high-grade EC with ambiguous features (n = 13, 33%) and low-grade endometrioid carcinoma (n = 1, 3%). HER2 IHC results were 0 or 1+ (n = 2, 5%), 2+ (n = 17, 46%), 3+ (n = 18, 49%), with HER2 heterogeneity in 15 cases. By FISH, the HER2/CEP17 ratio was ≥2.0 in 34 (92%) and &lt; 2.0 in 3 (8%); HER2 copy number (CN) was ≥6.0 in 25 (68%) and &lt; 6.0 in 12 (32%). HER2 copy number ≥6.0 was significantly correlated with HER2 amplification by NGS ( p&lt; 0.001). 18 (49%) patients had a co-existing somatic PIK3CA mutation. Amongst parameters assessed, only higher level of HER2 amplification by NGS was significantly associated with clinical benefit ( p= 0.036). None of these features were associated with progression-free survival. Notably, 2 patients with IHC 0/1+ had PD and 1 patient with IHC 3+, but non-amplified by FISH, achieved PR. Conclusions: In our cohort of EC patients treated with anti-HER2 therapy, higher level of HER2 amplification was associated with clinical benefit. Further studies with larger sample sizes are needed to determine whether any additional characteristics are associated with treatment response and survival.

Altered expression of Notch signaling, Tlr receptors, and surfactant protein expression after prostaglandin inhibition may be associated with the delayed labor in LPS-induced mice.

This study aims to investigate whether indomethacin (IND) delays preterm birth by regulating the Notch pathway, Tlr receptors, and Sp-A in the placenta in lipopolysaccharide (LPS)-induced preterm labor (PTL) model. CD-1 mice were distributed to the pregnant control (PC), Sham, PBS, IND (2 mg/kg; i.p.), LPS (25 μg/100 μl; intrauterine), and LPS + IND groups. The injections were performed on day 14.5 of pregnancy. Placentae were collected on day 15.5 of pregnancy, and immunohistochemical analyzes were performed. Differences in staining intensities between the Cox-1, Notch-1 (N1), Dll-1, Jagged-2 (Jag-2), Tlr-2, and Tlr-4 proteins were compared. Preterm labor rates were 100% and 66% (preterm delivery delayed 5 h) in the LPS and LPS + IND groups, respectively. In LPS-treated mice, a general morphological deterioration was observed in the placenta. Total placental mid-sagittal measurement was significantly reduced in the LPS-treated group, while it was similar to the PC group in the LPS + IND group. Cox-1 expression in the LZ increased, and Sp-A expression decreased after LPS injection, and IND administration diminished this increase. N1 expression increased in the labyrinth zone (LZ) and the junctional zone (JZ). Dll-1 and Jag-2 expression increased in the JZ after LPS injection (p < 0.0001). IND administration diminished Tlr-2 expression in the LZ and Tlr-4 expression in the JZ after LPS injection. In conclusion, PG (prostaglandin) inhibition may alter Notch signaling, Tlr, and Sp-A protein expression and may be associated with delayed labor in LPS-induced mice.

Characterization of chloride intracellular channel 4 in the regulation of human trophoblast function.

Proper placentation requires well controlled extravillous trophoblast cell (EVT) migration and invasion. Transforming growth factor β (TGFβ) signaling has been well characterized as negatively regulating EVT migration and invasion. CLIC4 is an enhancer of TGFβ signaling, however CLIC4's function in placentation and its association to placental TGFβ signaling is unknown. Here we aimed to investigate the role of CLIC4 on trophoblast cell function and its relationship to TGFβ signaling. CLIC4 was immunolocalized in human placenta throughout gestation and the first trimester decidua. siRNA was used to knockdown CLIC4 in a human trophoblast cell line (HTR8/SVneo) to reveal functional consequences of CLIC4 loss on cell adhesion, proliferation, migration and invasion via xCELLigence. qPCR was used to identify downstream targets of CLIC4 in HTR8/SVNeo cells. CLIC4 was widely expressed in the syncytiotrophoblast, cytotrophoblast and decidual cells across all trimesters of pregnancy with no significant difference in staining intensity in the different cellular compartments both across gestation and between compartments. Using immunofluorescent co-localization of CLIC4 and EVT marker HLA-G, we confirmed that CLIC4 localized to the cytoplasm of cell column EVTs in the first trimester decidua and nuclei of some EVTs that invaded in the decidua. Knockdown of CLIC4 in HTR8/SVneo cells significantly elevated cell adhesion, migration and invasion. Analysis of TGFβ signaling downstream targets identified that CDH2 and BAMBI expression were significantly increased after CLIC4 knockdown in HTR8/SVneo cells. Our data support an inhibitory role for CLIC4 in regulating trophoblast migration and invasion, likely acting in part via BAMBI and CDH2.

Diversity of complement activation in different thyroid diseases

ObjectiveTo investigate complement components expression in both thyroid tissues and serum from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD), and papillary thyroid cancer (PTC). MethodsC1q, mannose binding lectin (MBL), Bb, C4d, C3d and membrane attack complex (MAC) (C5b-9) deposition and complement regulate proteins (CD46, CD55 and CD59) expression in thyroid tissues from HT, GD, PTC, and control groups were examined by IHC. C1q, MBL, Bb, C4d, C3a, and soluble C5b-9 (sC5b-9) serum levels in the HT, GD, PTC, and healthy donor (HD) groups were measured by ELISAs. ResultsMAC deposition was detected in thyroid tissues in the HT, GD and PTC groups, but not the control group. MBL, Bb, C4d, C3d and MAC staining intensities in thyroid tissues were significantly higher in the HT and PTC groups than in the control group (all P < 0.05). The C1q level was higher in HT tissues than in control tissues (both P < 0.05). No complement component had a significant difference in staining intensities between the GD and control groups. CD55 and CD59 expression levels in thyroid tissues were higher in the PTC group than in the HT, GD and control groups (all P < 0.05). Similarly, CD46 levels were higher in HT tissues than in control tissues. Bb, C4d, C3a and sC5b-9 serum levels were significantly increased in HT, GD and PTC patients compared with HDs (all P < 0.05). ConclusionComplement is overactivated in HT and PTC, but not in GD. All the three pathways are activated in HT, and the MBL and alternative complement pathways are activated in PTC. These distinct complement activation profiles may participate in HT, GD and PTC pathogenesis.

Evaluation of immunohistochemical expression of E-cadherin in pleomorphic adenoma and adenoid cystic carcinoma

Background:Pleomorphic adenoma (PA) and adenoid cystic carcinoma (ADCC) are benign and malignant salivary gland tumors, respectively, with distinct behavior. They have similar origins and cell components. E-cadherins are the main homophilic cell adhesion molecules, which play a central role in maintaining epithelial integrity, functioning in intercellular adhesion and differentiation. Hence, changes in E-cadherin function are reflected in the morphologic events associated with the cellular arrangement, movement and wound healing.Aim:To study and compare the expression of E-cadherin immunostaining in PA and ADCC.Materials and Methods:Fifteen cases of each PA and ADCC were immunohistochemically stained with E-cadherin. Five cases of normal salivary gland tissues were taken as the positive control.Mann–Whitney U-test was used for statistical analysis.Results:About 86.6% of PA cases showed homogeneous staining. 66.6% of cases of ADCC showed heterogeneous staining. PA, cribriform and tubular patterns of ADCC predominantly showed moderate immune-staining and solid patterns of ADCC exhibited predominantly mild immunostaining. Depending on the intensity of staining, we found a significant P value between PA and the solid variant of ADCC.Conclusion:E-cadherin proved to be a better marker for epithelial phenotypes in PAs. In ADCC difference in staining intensity between different histological subtypes suggests that further studies should be done to assess the usefulness of an immuno-marker to know the aggressive behavior of ADCC.

Immunohistochemical Characterization of H3K4Me3 in Reactive Lymph Nodes and Follicular Lymphoma

Abstract Introduction/Objective Genes involved in histone methylation are frequently mutated in non-Hodgkin’s lymphomas. For instance, frequent mutations in genes encoding histone methytransferases MML2 and EZH2 are present in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). The aim of this study was to characterize the immunohistochemical expression of H3K4Me3 in benign/reactive lymph nodes (LNs) with comparison to follicular lymphoma (FL). Methods/Case Report Immunohistochemical staining with an anti-H3K4Me3 antibody was performed on FFPE whole slide section from patients with benign/reactive LNs (n=21), low grade (grade 1-2) FL (n=21). H3K4Me3 reactivity was scored for staining intensity and percentage of lymphocytes showing reactivity. Results (if a Case Study enter NA) The majority of the reactive LN sections (15 out of 21 cases) showed a distinct distribution of H3K4Me3 staining, with the majority of cells in the mantle zone and the interfollicular zones showing moderate-strong staining, whereas reactive germinal centers (GCs) showed significantly decreased or close to negative staining. Neoplastic follicles in all the FL cases contained positive cells with significantly stronger staining compared to that in the germinal centers in benign lymph nodes. The interfollicular zones, while diminished in FL due to expanded neoplastic follicles, showed retained H3K4Me3 staining. The difference in staining intensity between follicles and mantle/interfollicular zones became indistinct in FL. Conclusion H3K4Me3 expression in benign/reactive LNs is characterized by positive expression in lymphocytes in interfollicular and mantle zones and significantly decreased in GCs. However, the expression pattern is different in FL, which showed significantly increased expression in the follicles compared to that in reactive GCs. In FL, the expression in GCs is similar to that in the interfollicular and mantle zone lymphocytes. It is reported that about 90% of FL have MLL2 mutation and MLL2 is the enzyme responsible for the methylation of H3K4. So far, it is unclear whether the mutation of MLL2 in FL affects the overall methylation activity of the enzyme. In our study, there is increased methylation of H3K4 in the follicles of FL, which raises the possibility that MLL2 mutation somehow increases H3K4 methylation.

INDOMETHACIN CAN PROTECT PLACENTAL INFLAMMATION AND DELAY PRETERM BIRTH IN THE LPS-INDUCED PRETERM DELIVERY MODEL

The major cause of prematurity is preterm birth (PTB), associated with intrauterine inflammation. Defects in the Notch pathway harm placentation, and there is evidence between Notch activation and the inflammatory environment. In the action of PTB, surfactant A (SP-A) may have a pro-inflammatory or anti-inflammatory effect, and increased synthesis of prostaglandins illustrates their crucial roles in gestational tissues at parturition. Altogether, the potential of SP-A and prostaglandin inhibitors to prevent PTB through the placenta is worth exploring. This study evaluates the preventive effect of SP-A and Indomethacin (IND) treatment on placental inflammation in the LPS-induced PTB model. Forty-eight female CD-1 mice were distributed to pregnant control (PC), Sham, PBS, IND (2 mg/kg; intraperitoneally), LPS (25μg/100μl; intrauterine), LPS+IND, SP-A block (SP-A B; 20ug/100μl; intrauterine) groups. The injections were performed on day 14.5 of pregnancy. Placentae were removed on day 15.5 of pregnancy, and immunohistochemical analyzes were performed. Differences in staining intensities between the groups for Cox-1, Notch-1 (N1), Dll-1, Jagged-2 (Jag-2), SP-A, Tlr-2, and Tlr-4 proteins were compared using ANOVA and Sidak’s Multiple Comparison test. P values <0.05 were considered statistically significant. PTB rates were; 100%, 66% (in this group, delivery delayed for about 5 hours), and 50% in LPS, LPS+IND, SP-A B groups, respectively. LPS application caused damage to fetal and maternal vascular structures in the placenta, especially in the labyrinth zone (LZ). Placental volume decreased, and lymphocyte infiltration was observed. The morphological distinction between the compartments was unclear. N1 expression increased in both the junctional zone (JZ) and LZ. Cox-1 expression in the LZ decreased significantly (p<0.05), while the expression of N1, Dll- 1, and Jag-2 increased significantly (p<0.05). Tlr-2 and Tlr-4 expression increased significantly in LZ and JZ, respectively. In the LPS+IND group, the LZ morphology was similar to the control, and placenta zone boundaries were distinguishable. In the LPS+IND group, N1, Jag-2, and Tlr-4 expression decreased significantly (p<0.05). In the SP-A B group, Cox-1 expression increased significantly (p<0.05). In the PTB model, Notch signaling, SP-A, and prostaglandin-associated signaling are disturbed in the maternal-fetal exchange site, the LZ and hormonal production site, the JZ of the placentae. While SP-A modulates the LPS-induced inflammatory response related to PTB, IND can prevent PTB via decreasing inflammation in the LZ.

Loss of CD22 expression and expansion of a CD22dim subpopulation in adults with relapsed/refractory B-lymphoblastic leukaemia after treatment with Inotuzumab-Ozogamicin

Treatment options for relapsed or refractory B-lymphoblastic leukaemia (r/r B-ALL) are limited and the prognosis of these patients remains dismal, but novel immunotherapeutic options such as the anti-CD22 antibody–drug-conjugate Inotuzumab-Ozogamicin (InO) have improved outcomes in these patients. Flow cytometry is essential to assess antigen-expression prior to treatment initiation of antigen-directed immunotherapies. Here, we present flow cytometric and clinical data of three adult patients with r/r B-ALL who failed treatment with InO associated with reduced or lost antigen-expression. In addition, we present comparative data on two different diagnostic CD22-specific antibody clones that exhibit significant differences in staining intensities.

Changes in qualitative and quantitative traits of birch (Betula pendula) pollen allergenic proteins in relation to the pollution contamination

Birch (Betula pendula) pollen causes inhalant allergy in about 20% of human population in Europe, most of which is sensitive to the main birch allergen, Bet v1. The aim of the study was to find out (i) whether and how the analysed birch individuals differ in regard to composition of individual subunits of pollen proteins and to protein content in these subunits; (ii) whether the level of particulate matter relates to concentration of Bet v1 allergen. Study was performed in Southern Poland, in 2017–2019. Pollen material was collected at 20 sites, of highly or less polluted areas. Protein composition was analysed by SDS-PAGE, while the concentration of Bet v1 was evaluated by ELISA. The obtained results were estimated at the background of the particulate matter (PM10) level and the birch pollen seasons in Kraków. The electrophoregrams of pollen samples collected at different sites showed huge differences in staining intensities of individual protein subunits, also among important birch allergens: Bet v1, Bet v2, Bet v6 and Bet v7. The level of Bet v1 was significantly higher in the pollen samples collected at the more polluted sites. While the birch pollen allergenic potential is determined, the both pollen exposure and the content of the main allergenic components should be considered, as factors causing immunological response and clinical symptoms manifestation in sensitive individuals.

Effect of maternal overnutrition on predisposition to insulin resistance in the foal: Maternal parameters and foal pancreas histoarchitecture

Results from previous studies indicate that maternal overnutrition during late gestation predisposes foals to metabolic disease, however, specific mechanisms resulting in disease remain unknown. Quarter Horse mares (n = 16), were randomly assigned to dietary treatments, beginning on gestational day 235, and consisted of a control group (CON- diet meeting nutrient requirement; n = 8) or an overfed diet (HIGH; n = 8) where mares received an additional 40 % above CON. On gestational days 285 and 315, an intravenous glucose tolerance test (FSIGTT) was conducted. Following parturition, foals were separated from the mare, prohibited from nursing, and an FSIGTT was conducted at 2 h postpartum. Foals were immediately euthanized and tissues preserved for analyses. There was no effect of treatment on foal BW (P = 0.50), pancreas weight (P = 0.60), or FSIGTT area under the curve for glucose (P = 0.80) and insulin (P = 0.70). Colocalization of α-amylase to isolate pancreatic islets of Langerhans indicated increased islet number and size in foals from HIGH mares (P < 0.01). Immunofluoresent analysis of insulin, glucagon, and somatostatin indicate no difference in intensity of staining (P> 0.10). Foals exposed to overnutrition during peak fetal growth had altered pancreatic islet development that may lead to adult-onset metabolic disease.

Intradiscal Delivery of Anabolic Growth Factors and a Metalloproteinase Inhibitor in a Rabbit Acute Lumbar Disc Injury Model.

The purpose of our study was to examine the effect of controlled delivery of TGF-β3, BMP-4, and TIMP-2 with a biocompatible biopolymer, chitosan, on an acutely injured intervertebral disc (IVD) in a rabbit model. After conducting an in vitro analysis of the chondrogenic capacity of the biomolecule cocktail use (ie, TGF-β3, BMP-4, and TIMP-2) and confirming stem cell viability in chitosan hydrogel, 15 New Zealand white rabbits underwent a lateral approach of the L1 to L4 IVDs. In each rabbit, the L2 to L3 IVD was left pristine, whereas the L1 to L2 and the L3 to L4 IVDs in each rabbit underwent nucleotomy via a 25-G needle, and the animal was subsequently randomized to no further treatment (defect only), chitosan alone, Chitosan + TGF-β3 + BMP-4, or chitosan + TGF-β3 + BMP-4 + TIMP-2. At 6 weeks after injury and intervention, the rabbits were killed and spines harvested to undergo quantitative T2 magnetic resonance imaging (MRI) and subsequent histologic analysis. In the in vitro analysis, cells treated with experimental media containing TGF-β3, BMP-4, and TIMP-2 exhibited staining indicative of GAG production and began to exhibit a chondrocytic morphology. Quantitative T2 MRI mapping demonstrates that discs treated with chitosan, chitosan containing TGF-β3 and BMP-4, or chitosan containing TGF-β3, BMP-4, and TIMP-2 had consistently higher T2 relaxation times compared with defect-only discs. When the T2 relaxation times of each treatment group and defect-only discs were normalized to the healthy control disc, it was found that the T2 relaxation time of discs treated with chitosan containing TGF-β3 and BMP-4 and discs treated with chitosan containing TGF-β3, BMP-4, and TIMP-2 were significantly greater compared with defect-only discs (P = .048 and P = .013, respectively). Histologically, animals that received chitosan only, or chitosan with TGF-β3 and BMP-4, showed a significantly higher intensity of Safranin-O staining (P = .016 and P = .02, respectively) compared with control discs, whereas the difference in staining intensity in animals that received chitosan loaded with TGF-β3, BMP-4, and TIMP-2 failed to achieve significance (P = .161). A combination of chitosan, TGF-β3, and BMP-4 was effective at promoting regeneration in an acute disc injury rabbit model, whereas TIMP-2 did not have a significant effect.

QuPath. A new digital imaging tool for geropathology.

Translational research regularly utilizes immunohistochemistry (IHC) to investigate pathological differences in the clinical or laboratory setting. However, the majority of these studies require the extensive work of a trained pathologist to analyze slides in a meaningful way. In order to explore new ways to quantitate IHC stains in a manner that is reproducible and efficient for both pathologists and research scientists, QuPath was explored as a new digital imaging tool. The hippocampal area of brains from older sleep deprived mice were stained using established IHC protocols to explore biomarker levels that would be insightful for measurable differences. Application features of QuPath are described that quantitatively show sleep deprived mice had robust differences in staining intensity for four different biomarkers compared to non-sleep deprived mice. These observations provide the rationale for QuPath as a digital imaging tool to enhance the quantitative and qualitative usefulness of IHC staining in the field of geropathology.

Detection of circulating anti-retinal antibodies in dogs with sudden acquired retinal degeneration syndrome using indirect immunofluorescence: A case-control study

Sudden acquired retinal degeneration syndrome (SARDS) in dogs is proposed to have an immune-mediated etiology. However, there is conflicting evidence regarding the presence of antiretinal antibodies, as assessed by western blotting, in the serum of SARDS patients. Because of the possibility that antibodies recognize only conformational epitopes, we hypothesized that a more sensitive method to investigate circulating retinal autoantibodies in SARDS is immunofluorescence. Sera from 14 dogs with early SARDS, and 14 age- and breed-matched healthy control dogs were screened for circulating antiretinal IgG, IgM, IgE and IgA using indirect immunofluorescence on lightly fixed frozen sections of normal canine retina. Controls without canine serum were also performed. A nuclear counterstain was used to identify cellular retinal layers. Images were obtained using a fluorescence microscope, and 2-3 separate masked observers graded retinal layers for fluorescence staining intensity using a 0–3 scale. Total circulating IgG and IgM was assessed by radial immunodiffusion. Statistical analysis was performed using 2-way ANOVA, paired 2-tailed student's t-test and correlation analysis. Intensity of IgG staining of photoreceptor outer segments was significantly higher using serum from dogs with SARDS compared with healthy controls in 2/3 observers (P < 0.05). Intensity of IgM staining throughout the retina was higher in SARDS dogs compared to matched healthy controls (P < 0.0001), although no specific retinal layer was statistically significant. There were no differences in staining intensity for IgE or IgA. Dogs with SARDS had a comparably lower circulating IgG and higher IgM than healthy controls (P = 0.01 and 0.001 respectively) and IgG and IgM were negatively correlated (r = −0.69, P = 0.007). Despite having decreased serum IgG compared with healthy controls, circulating IgG in dogs with SARDS binds photoreceptor outer segments to a greater extent. Dogs with SARDS have a relatively higher circulating IgM than matched healthy controls. The pathogenic nature of these antibodies is unknown.

Abstract C017: Signaling through Ref-1 and STAT3 in soft tissue sarcoma (MPNST) and the effects of perturbing this pathway on tumor cell survival and gene expression

Abstract Malignant Peripheral Nerve Sheath Tumor (MPNST) is a rare soft tissue sarcoma that can arise from patients with NF1 (neurofibromatosis type 1). These patients are at a much greater risk of developing MPNST than the general population (10% vs. 0.01%, respectively). Existing chemotherapeutic and targeted agents have thus far not been successful in MPNST treatment, with a 5-year patient survival rate of just 35%-50%. Recent research implicates Signal Transducer and Activator of Transcription-3 (STAT3) and Hypoxia Inducible Factor 1 (HIF1) in driving MPNST. STAT3 is a transcription factor implicated in several cancers, and phosphorylated STAT3 (p-STAT3) expression indicates aggressive disease at disease onset in MPNST. STAT3 is activated upon phosphorylation by Janus Kinase (JAK), but its DNA binding and transcriptional activity is regulated by Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1 or Ref-1) redox function. Ref-1 is a multifunctional protein involved in repairing DNA damage via endonuclease activity and in redox regulation of various transcription factors including STAT3, HIF-1α, and NFκB. High expression levels of Ref-1 indicate decreased survival in several cancers. We characterized Ref-1 and p-STAT3 expression in several MPNST samples including: the IN Pediatric BioBank, and established MPNST cell lines, new patient-derived tumor cells, and genetically engineered mouse models. We observed strong Ref-1 and phospho-STAT3 staining in the majority of these samples. We examined the expression of p-STAT3 and Ref-1 in mice that have conditional ablation in the Schwann cells of Nf1-/-; Arf+/- and Nf1-/-; Arf-/- mice and found a significant increase in positivity of p-STAT3 and Ref-1 expression in the sections containing MPNST. It also appears that there may be differences in staining intensity based on Arf genotype as the Nf1-/-; Arf-/- have stronger staining than the Nf1-/-; Arf+/-. Knocking down Ref-1 or STAT3 impairs MPNST growth in vitro. Inhibiting the redox activity of Ref-1 using the redox-specific APE1 inhibitor, APX3330 (and its next generation analogs), or inhibiting STAT3 activity using Napabucasin also resulted in reduced in vitro growth. Investigation into the apoptotic pathways that are activated following treatment demonstrate activation of caspase 3/7. Several biomarkers downstream of Ref-1 and STAT3 were downregulated following Ref-1 redox or STAT3 inhibition. We also performed RNA-sequencing to identify differentially expressed genes (DEGs) following Ref-1 or STAT3 knockdown and implicate new targets associated with clinical response in MPNST. In vivo experiments performed on mice implanted with ST88-14 cells using either APX2009 (APX3330 analog) or Napabucasin both resulted in significantly reduced tumor growth and additional combination studies are underway. Obtaining these results with clinically-tested Ref-1 and STAT3 inhibitors make the translation of this work highly plausible for pediatric patients with MPNST in the future. We will use both PDX and genetically engineered models of MPNST to test and validate these novel targets. Citation Format: Fenil Shah, Olivia Babb, Chi Zhang, Silpa Gampala, Emily Zhang, Steven D Rhodes, Andrew R. Tee, Brian Calver, Ellie Rad, Verena Staedtke, Karen E Pollok, D. Wade Clapp, Mark R. Kelley, Melissa L. Fishel. Signaling through Ref-1 and STAT3 in soft tissue sarcoma (MPNST) and the effects of perturbing this pathway on tumor cell survival and gene expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C017. doi:10.1158/1535-7163.TARG-19-C017

TdT expression in germ cell tumours: a possible immunohistochemical cross-reaction and diagnostic pitfall

AimsVery recent papers proposed a possible role for the expression of terminal deoxynucleotidyl transferase (TdT) in the tumourigenesis of gonadal and extragonadal germ cell-derived tumours (GCTs). Our multicentric study evaluated...