Protein Immunostaining Research Articles

Comparison of background characteristics and neuropathology findings between medico-legal autopsy cases with traumatic axonal injury, vascular axonal injury, or absence of axonal injury in β-amyloid precursor protein stain.

In forensic neuropathology, the β-amyloid precursor protein (β-APP) immunostain is used to diagnose axonal injury (AI). The two most common aetiologies are traumatic (TAI) and ischaemic (vascular; VAI). We aimed to identify background characteristics and neuropathology findings that are suggestive of TAI, VAI, or no AI in neuropathologically examined medico-legal autopsy cases. The dataset comprised 166 cases from Finland over the period 2016-2023. The diagnosis of AI was based on β-APP stain (TAI, VAI, or no AI). Data on background characteristics and neuropathology findings were collected from cause-of-death investigation documents. Prevalence ratios were calculated for each variable to enable comparisons between the AI categories. The sample were 71.7% males; median age was 41 years (range 0-96). There were 26 cases with TAI, 44 with VAI, and 96 with no AI. The variables that showed statistical significance and had at least two-fold prevalence among TAI cases compared to VAI cases were: a documented recent injury; and presence of any extracranial/cranial/intracranial injury (including subdural haemorrhage [SDH], subarachnoid haemorrhage [SAH], intracerebral/ventricular haemorrhage [ICVH], or contusion) in autopsy or neuropathology. Correspondingly, variables indicating TAI over no AI were: a documented recent injury; postinjury survival ≥ 24h; and presence of any extracranial/cranial/intracranial injury (including SDH, SAH, ICVH, contusion), herniation, or infarction in autopsy or neuropathology. Postinjury survival < 30min was identified as an indicator of no AI over TAI. Finally, variables indicating VAI over no AI were: postinjury survival ≥ 24h; lack of external injury to the head; and presence of SDH, brain oedema, herniation, or infarction in autopsy or neuropathology. In conclusion, we report several differences in characteristics and findings between cases diagnosed with TAI, VAI, and no AI. Our findings may help estimate the likelihood and potential aetiology of AI based on background characteristics and other neuropathology findings.

Biological Behavior of Bioactive Glasses SinGlass (45S5) and SinGlass High (F18) in the Repair of Critical Bone Defects.

This study evaluated the osteogenic potential of the bioactive glasses SinGlass (45S5) and SinGlass High (F18) in regenerating critical bone defects in rat calvaria. Both biomaterials promoted new bone formation around the particles, with the SinGlass High (F18) group exhibiting a higher rate of bone maturation. Histomorphological and birefringence analyses revealed better organization of the newly formed bone in the biomaterial-treated groups, and immunohistochemistry indicated the expression of osteogenic markers such as osteocalcin, immunostaining for bone morphogenetic protein 2 (BMP 2), and immunostaining for bone morphogenetic protein 4 (BMP 4). Microtomography computadorized (Micro-CT) revealed centripetal bone formation in both groups, with greater integration of the particles into the surrounding bone tissue. The superior performance of SinGlass High (F18) was attributed to its higher potassium and magnesium content, which enhance osteoconductivity. After 42 days, the SinGlass High (F18) group showed the highest percentage of new bone formation, in line with previous studies. Although our results are promising, the limited follow-up period and use of a single animal model highlight the need for further research to validate clinical applicability. SinGlass High (F18) appears to be a viable alternative to autografts in bone repair, with potential to improve tissue integration and accelerate recovery.

Feline Papillomatosis.

Different types of feline papillomaviruses (PVs) are associated with a variety of skin lesions and neoplasia, such as papillomas and cell carcinomas, but the virus can also be found in healthy skin. In this review, the European Advisory Board on Cat Diseases (ABCD), a scientifically independent board of veterinary experts on feline infectious diseases from 11 European Countries, discusses the current knowledge of feline PV infections. Cats most likely become infected through lesions or abrasions of the skin. Most PV infections remain asymptomatic. Besides cat-specific PVs, DNA sequences most closely related to human and bovine PVs have been detected in feline skin lesions. Diagnosis is supported by the histological detection of PV-induced cell changes and intralesional detection of viral antigen (immunostaining) or viral DNA (in situ hybridization). Immunostaining of p16CDKN2A protein (p16) can be performed as a proxy marker for PV-induced neoplasms. There is no specific treatment for PV-induced skin lesions. Spontaneous regression commonly occurs. In the case of invasive squamous cell carcinoma (ISCC), complete excision should be considered, if possible.

Microglial activation without peripheral immune cell infiltration characterises mouse and human cerebral small vessel disease.

Cerebral small vessel diseases (SVDs) involve diverse pathologies of the brain's small blood vessels, leading to cognitive deficits. Cerebral magnetic resonance imaging (MRI) reveals white matter hyperintensities (WMHs), lacunes, microbleeds and enlarged perivascular spaces in SVD patients. Although correlations of MRI and histopathology help to understand the pathogenesis of SVD, they do not explain disease progression. Mouse models, both genetic and sporadic, are valuable for studying SVD, but their resemblance to clinical SVD is unclear. The study examined similarities and differences between mouse models of SVDs and human nonamyloid SVD specimens. We analysed four mouse models of SVD (hypertensive BPH mice, Col4a1 mutants, Notch3 mutants and Htra1-/- mice) at different stages for changes in myelin, blood-brain barrier (BBB) markers, immune cell populations and immune activation. The observations from mouse models were compared with human SVD specimens from different regions, including the periventricular, frontal, central and occipital white matter. Postmortem MRI followed by MBP immunostaining was used to identify white matter lesions (WMLs). Only Notch3 mutant and hypertensive BPH mice showed significant changes in myelin basic protein (MBP) immunostaining, correlating with MRI patterns. These changes were linked to altered microglial morphology and focal plasma protein staining around blood vessels, without peripheral immune cell infiltration. In human specimens, both normal-appearing white matter (NAWM) and WMLs lacked peripheral cell infiltration. However, WMLs displayed altered microglial morphology, reduced myelin staining and occasional fibrinogen staining around arterioles and venules. Our data show that Notch3 mutants and hypertensive BPH/2J mice recapitulate several features of human SVD, including microglial activation, focal sites of demyelination and perivascular plasma protein leakage without peripheral immune cell infiltration.

Abstract B026: Glioblastoma multiforme disseminates outside the central nervous system

Abstract Introduction: The liquid biopsy (LB) concept has gained significant attention in the last decade and has the potential to revolutionize cancer care. However, more validation in clinical trials is required on the value of LB in medical settings. Here, we focus on glioblastoma multiforme (GBM), a highly invasive and fatal subtype of the central nervous system malignant tumors. Circulating tumor cells (CTCs) are primary tumor cells released into the peripheral blood and migrating to distant organs. CTC presence is related to tumor spread and recurrence. In GBM, factors like short patient survival, the blood-brain barrier (BBB), and lack of lymphatic drainage in brain tissue make systemic dissemination rare but possible. Thus, noninvasive CTC detection can hold potential for diagnostic, predictive, and prognostic applications. Material and Methods: For cell stability preservation before sample processing, the peripheral blood samples from curatively resected GBM patients were collected preoperatively in Cell-Free DNA BCT® tubes (Streck, Inc.). CTCs were identified using CytoTrack CT11TM (2/C), a semi-automated immunofluorescence microscopy using glial fibrillary acidic protein, vimentin, and CD45 immunostaining, and DAPI counterstaining. The preliminary Kaplan-Maier survival analysis based on CTC positivity was performed in 39 patients with a follow-up longer than two years. Results: We analyzed the CTC presence in 100 GBM patient peripheral blood samples. The CTCs or cancer cell clusters were found in 35% of GBM patients. The Kaplan-Maier survival analysis did not show a correlation between CTC presence and survival. This phenomenon may be associated with the generally short survival of these patients, which is expected to increase with advances in oncology and palliative care and/or a possible suppression of tumor cell growth outside the CNS. Conclusions: We have observed the CTCs in the peripheral blood of GBM patients. The patient enrollment and clinical data recording are still ongoing. Citation Format: Pavel Stejskal, Josef Srovnal, Alona Rehulkova, Ondrej Kalita, Marek Slachta, Marian Hajduch. Glioblastoma multiforme disseminates outside the central nervous system [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B026.

Evaluation of a Novel MET-Targeting Camelid-Derived Antibody in Head and Neck Cancer.

In head and neck squamous cell carcinoma (HNSCC), the mesenchymal epithelial transition (MET) receptor drives cancer growth, proliferation, and metastasis. MET is known to be overexpressed in HNSCC and, therefore, is an appealing therapeutic target. In this study, we evaluated MET expression in patients with HNSCC and investigated the potential imaging application of a novel MET-binding single-domain camelid antibody using positron emission tomography/computed tomography (PET/CT) in a preclinical MET-expressing HNSCC model. Multiplex immunostaining for MET protein performed on a tissue microarray from 203 patients with HNSCC found 86% of patients to have MET expression, with 14% having high expression and 53% having low MET expression. Using The Cancer Genome Atlas (TCGA) database, high MET RNA expression was associated with worse progression-free survival and overall survival in patients with HPV-negative HSNCC. Utilizing flow cytometry and immunofluorescence, our novel camelid antibody fused to a human IgG Fc chain (1E7-Fc) showed high binding affinity and specificity to high MET-expressing Detroit 562 cells but not to low MET-expressing HNSCC cells. The efficacy and biodistribution of [89Zr]Zr-1E7-Fc as a PET imaging agent was then investigated in a MET-expressing head and neck xenograft model. [89Zr]Zr-1E7-Fc rapidly localized and showed high tumor uptake in Detroit 562 xenografts (8.4% ID/g at 72 h postinjection), with rapid clearance from the circulatory system (2.7 tumor-to-blood radioactivity ratio at 72 h postinjection). Our preclinical data suggest that the camelid antibody 1E7-Fc could be a potential theranostic agent for HNSCC. Further investigations are warranted to confirm these findings in patients and to evaluate 1E7-Fc as an imaging agent and platform to deliver radionuclide or drug therapy to MET-driven cancers.

A powerful and versatile new fixation protocol for immunostaining and in situ hybridization that preserves delicate tissues

BackgroundUnderstanding how genes function to heal wounds and restore lost tissue is essential for studying regeneration. Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression patterns of genes in different biological systems. Yet, existing methods to permeabilize samples for WISH can damage or destroy fragile regenerating tissues, thereby preventing such experiments.ResultsHere, we describe a new protocol for in situ hybridization (ISH) and immunostaining in the highly regenerative planarian Schmidtea mediterranea. This new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both the assays and prevents degradation of the epidermis and regeneration blastema. The NAFA protocol achieves this without the use of proteinase K digestion which likely leads to better preservation of antigen epitopes. We show that the NAFA protocol successfully permits development of chromogenic and fluorescent signals in situ, while preserving the anatomy of the animal. Furthermore, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Additionally, the tissue fixation protocol was easily adapted for regenerating killifish tail fin, which yielded better ISH signal with minimal background.ConclusionsThus, the NAFA protocol robustly preserves the delicate wounded tissues while also facilitating probe and antibody penetration into internal tissues. Furthermore, the fixation protocol is compatible for WISH on regenerating teleost fins suggesting that it will be a valuable technique for studying the processes of wounding response and regeneration in multiple species.

Systemic Inflammation Decreases Initial Brain Injury but Attenuates Neurite Extension and Synapse Formation during the Repair of Injured Brains.

In this study, we explored the impact of systemic inflammation on initial brain injury and repair processes, including neurite extension and synapse formation. For this purpose, we established a brain injury model by administering adenosine triphosphate (ATP), a component of damage-associated molecular patterns (DAMPs), through stereotaxic injection into the striatum of mice. Systemic inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS-ip). Bulk RNA-sequencing (RNA-seq) analyses and immunostaining for microtubule-associated protein 2 (MAP2) and tyrosine hydroxylase (TH) showed that LPS-ip led to a reduction in initial brain injury, but inhibited neurite extension into the damaged brain. LPS-ip upregulated expression of defense response genes and anti-apoptotic genes, but decreased expression of genes associated with repair and regeneration. In addition, LPS-ip reduced levels of vGlut1 and PSD95 (markers for excitatory pre and post synapses, respectively), but had little effect on vGAT and gephyrin (markers for inhibitory pre and post synapses, respectively). Taken together, these findings suggest that systemic inflammation reduce initial damage but impede subsequent repair process.

Daikenchuto, a Japanese herbal medicine, ameliorates experimental colitis in a murine model by inducing secretory leukocyte protease inhibitor and modulating the gut microbiota.

Inflammatory bowel disease (IBD) is a refractory inflammatory disorder of the intestine, which is probably triggered by dysfunction of the intestinal epithelial barrier. Secretory leukocyte protease inhibitor (SLPI) secreted by colon epithelial cells protects against intestinal inflammation by exerting anti-protease and anti-microbial activities. Daikenchuto (DKT) is one of the most commonly prescribed Japanese traditional herbal medicines for various digestive diseases. Although several animal studies have revealed that DKT exerts anti-inflammatory effects, its detailed molecular mechanism is unclear. This study aimed to clarify the anti-inflammatory mechanism of DKT using a murine colitis model, and to evaluate its potential as a therapeutic agent for IBD. Experimental colitis was induced in wild-type (WT) mice and SLPI-deficient (KO) mice by dextran sulfate sodium (DSS) after oral administration of DKT. The resultant clinical symptoms, histological changes, and pro-inflammatory cytokine levels in the colon were assessed. Expression of SLPI in the colon was detected by Western blotting and immunohistochemistry. Composition of the gut microbiota was analyzed by 16S rRNA metagenome sequencing and intestinal metabolites were measured by gas chromatography-mass spectrometry analysis. Intestinal epithelial barrier function was assessed by oral administration of FITC-dextran and immunostaining of tight junction proteins (TJPs). Oral administration of DKT increased the number of butyrate-producing bacteria, such as Parabacteroides, Allobaculum, and Akkermansia, enhanced the levels of short-chain fatty acids, including butyrate, in the colon, induced SLPI expression, and ameliorated DSS-induced colitis in WT mice. We found that mouse colon carcinoma cell line treatment with either DKT or butyrate significantly enhanced the expression of SLPI. Moreover, supplementation of DKT protected the intestinal epithelial barrier with augmented expression of TJPs in WT mice, but not in KO mice. Finally, the composition of the gut microbiota was changed by DKT in WT mice, but not in KO mice, suggesting that DKT alters the colonic bacterial community in an SLPI-dependent manner. These results indicate that DKT exerts anti-inflammatory effects on the intestinal epithelial barrier by SLPI induction, due, at least in part, to increased butyrate-producing bacteria and enhanced butyrate levels in the colon. These results provide insight into the mechanism of the therapeutic effects of DKT on IBD.

SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue.

Postmortem human brain tissue is a critical resource for studying neurodegenerative disease, providing critical insights into cellular morphology, pathology, and network connectivity. To improve standard microscopy and enable high-resolution, three-dimensional (3D) images of tissues at the subcellular level, tissue-clearing methods have been developed. These 3D images allow for the analysis of large regions of interest and can be used to study structural and spatial changes that occur during neurodegeneration. Additionally, 3D imaging facilitates the visualization of whole-cell morphology, especially in cells with long processes that would otherwise be truncated in single-plane images. Human brain tissue is especially challenging for tissue clearing due to the abundance of lipids in myelin and the need for optimal fixation and low postmortem intervals. Formaldehyde-based fixatives, commonly used in preserving tissue, hinder antibody binding by crosslinking important antibody epitopes, and fluorescent microscopy requires the incorporation of fluorescent labels through passive diffusion or electrophoresis. Recent studies have focused on optimally fixed human brain tissue with short postmortem intervals, limiting the general applicability of these methods. To address these challenges, we developed SHARD (SHIELD, antigen retrieval, and delipidation), a simple and widely applicable method for clearing and labeling human brain tissue, which can be applied to long-term banked human brain tissue preserved in formaldehyde. SHARD is a novel addition to the SHIELD tissue clarification method, combining antigen retrieval, tissue clearing, and staining of 200-μm sections from long-term banked human brain tissue. The SHARD method is effective for postmortem intervals (PMIs) ranging from 10 to 72 h in multiple neurodegenerative diseases and control samples. In this study, we demonstrate that the SHARD method significantly enhances the immunostaining of glial fibrillary acidic protein (GFAP), an astrocytic cytoskeletal marker. Overall, the combination of antigen retrieval and tissue delipidation holds great potential for achieving detailed 3D immunostaining in long-term formaldehyde-fixed postmortem human brain tissue, opening new avenues for research and discovery.

Immunostaining for Mismatch Repair Complex Proteins Impacts on Clinical-Pathological Characteristics and Prognosis of Adenoid Cystic Carcinoma of Salivary Glands.

To evaluate the influence of MMR proteins on clinicopathological characteristics and prognosis of salivary gland adenoid cystic carcinoma (ACC). The solid pattern of ACC showed lower expression for MSH2 (p = 0.039). Significant imbalance in MSH2/MSH6 immunostaining was observed in all histological patterns (p < 0.001), and imbalance in PMS2/MLH1 immunostaining was observed in the cribriform pattern (p = 0.011). The presence of capsule was associated with high expression of MSH6 (p = 0.019), MLH1 (p = 0.045) and PMS2 (p = 0.009). The absence of cribriform pattern (p = 0.002) and capsule pattern (p = 0.025), as well as low expression for MSH6 (p = 0.006) and PMS2 (p = 0.037) were associated with lower overall survival. In multivariate analysis, loss of MSH2 (p = 0.039) and MLH1 (p = 0.017) were significantly associated with worse overall survival. Twenty-four ACC were clinical-pathologically evaluated and we perform immunohistochemistry for MSH2, MSH6, PMS2 and MLH1. Percentage counting of positive cells was performed in 10 fields of each histological pattern (cribriform, tubular and solid) and the averages of the 30 fields were considered for evaluation with other clinical-pathological variables (Kruskal-Wallis/Dunn, Friedman/Dunn, chi-square, Log-Rank Mantel-Cox tests and Cox regression; SPSS v20.0, p < 0.05). Salivary glands' ACC shows imbalance of the MMR complex and loss of expression of its components is associated with the overall survival of these patients.

Adipocytes Promote Endometrial Cancer Progression Through Activation of the SIRT1-HMMR Signaling Axis.

Adipocyte is a predominant component of the omental adipose tissue that influences the tumor microenvironment and increases the risk of endometrial cancer progression (EC), however, little is known about the underlying mechanism. In this study, using a co-culture model, we found that the adipocyte-EC cell interaction promoted SIRT1 signaling in vitro and in vivo xenograft mice models. Furthermore, immunostaining of SIRT1 protein showed significantly higher expression of SIRT1 in endometrial cancer patients than in normal endometria. RNA sequencing analysis revealed HMMR (hyaluronan-mediated motility receptor), an oncogene, as a downstream effector of SIRT1 in adipocyte-associated EC. Transient knockdown and chromatin immunoprecipitation assays showed that SIRT1 inhibition impedes transcription of the HMMR gene via FOXM1, and reduced expression of HMMR in co-cultured EC cells blocks AURKA activation via TPX2, leading to cell cycle arrest. This is the first study to report the positive correlation between SIRT1 and HMMR in EC patient tumors and might be used as a potential biomarker in EC. Notably, SIRT1 regulates HMMR expression in a FOXM1-dependent manner, and interfering with SIRT1 may provide a promising strategy for the management of endometrial cancer.

Longitudinal Imaging of Injured Spinal Cord Myelin and White Matter with 3D Ultrashort Echo Time Magnetization Transfer (UTE-MT) and Diffusion MRI.

Quantitative MRI techniques could be helpful to noninvasively and longitudinally monitor dynamic changes in spinal cord white matter following injury, but imaging and postprocessing techniques in small animals remain lacking. Unilateral C5 hemisection lesions were created in a rat model, and ultrashort echo time magnetization transfer (UTE-MT) and diffusion-weighted sequences were used for imaging following injury. Magnetization transfer ratio (MTR) measurements and preferential diffusion along the longitudinal axis of the spinal cord were calculated as fractional anisotropy or an apparent diffusion coefficient ratio over transverse directions. The area of myelinated white matter was obtained by thresholding the spinal cord using mean MTR or diffusion ratio values from the contralesional side of the spinal cord. A decrease in white matter areas was observed on the ipsilesional side caudal to the lesions, which is consistent with known myelin and axonal changes following spinal cord injury. The myelinated white matter area obtained through the UTE-MT technique and the white matter area obtained through diffusion imaging techniques showed better performance to distinguish evolution after injury (AUCs > 0.94, p < 0.001) than the mean MTR (AUC = 0.74, p = 0.01) or ADC ratio (AUC = 0.68, p = 0.05) values themselves. Immunostaining for myelin basic protein (MBP) and neurofilament protein NF200 (NF200) showed atrophy and axonal degeneration, confirming the MRI results. These compositional and microstructural MRI techniques may be used to detect demyelination or remyelination in the spinal cord after spinal cord injury.

Licochalcone A attenuates NMDA-induced neurotoxicity

ABSTRACT This study investigates the effect of Licochalcone A (Lico-A), a flavonoid from licorice roots known for its anti-inflammatory, anti-cancer, and antioxidant properties, on NMDA-induced neurotoxicity in primary cultured rat hippocampal neurons. The study measured cell survival following NMDA and Lico-A exposure, revealing that Lico-A at a 2.5 μg/ml significantly improved cell viability, countering the detrimental effects of NMDA. The study also analyzed synaptic changes by examining both postsynaptic density 95 (PSD95) and synaptophysin-targeted imaging, showing that Lico-A treatment resulted in a significant increase in synaptic puncta, contrasting with the reduction observed under NMDA exposure. Furthermore, levels of phosphorylated mixed lineage kinase domain-like pseudokinase (P-MLKL) and phosphorylated receptor-interacting serine/threonine-protein kinase 3 (P-RIP3), key necroptosis regulators, were measured using Western blotting. The results showed an increase in P-MLKL and P-RIP3 in neurons exposed to NMDA, which was reduced following Lico-A treatment. The response of astrocyte and microglia was also evaluated by immunostaining for glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (IBA-1) and tumor necrosis factor alpha (TNF-α). These markers exhibited heightened expression in the NMDA group, which was substantially reduced by Lico-A treatment. These findings suggest that Lico-A has neuroprotective effects against NMDA-induced neurotoxicity, potentially contributing to synaptic preservation, inhibition of neuronal necroptosis, and modulation of glial activation. Therefore, Lico-A shows promise as a neuroprotective agent for conditions associated with NMDA-related neurotoxicity.

Quantification of cardiac capillarization in basement-membrane-immunostained myocardial slices using Segment Anything Model

Decreased myocardial capillary density has been reported as an important histopathological feature associated with various heart disorders. Quantitative assessment of cardiac capillarization typically involves double immunostaining of cardiomyocytes (CMs) and capillaries in myocardial slices. In contrast, single immunostaining of basement membrane protein is a straightforward approach to simultaneously label CMs and capillaries, presenting fewer challenges in background staining. However, subsequent image analysis always requires expertise and laborious manual work to identify and segment CMs/capillaries. Here, we developed an image analysis tool, AutoQC, for automatic identification and segmentation of CMs and capillaries in immunofluorescence images of basement membrane. Commonly used capillarization-related measurements can be derived from segmentation results. By leveraging the power of a pre-trained segmentation model (Segment Anything Model, SAM) via prompt engineering, the training of AutoQC required only a small dataset with bounding box annotations instead of pixel-wise annotations. AutoQC outperformed SAM (without prompt engineering) and YOLOv8-Seg, a state-of-the-art instance segmentation model, in both instance segmentation and capillarization assessment. Thus, AutoQC, featuring a weakly supervised algorithm, enables automatic segmentation and high-throughput, high-accuracy capillarization assessment in basement-membrane-immunostained myocardial slices. This approach reduces the training workload and eliminates the need for manual image analysis once AutoQC is trained.

Inhibitory effect of all-trans retinoic acid on ferroptosis in BeWo cells mediated by the upregulation of heme Oxygenase-1

IntroductionThis study aimed to explore the association between ferroptosis, a newly identified type of cell death, and the role of retinoic acid in developing pregnancy complications. Therefore, the effects of all-trans retinoic acid (ATRA) on ferroptosis susceptibility in BeWo cells were assessed to understand abnormal placental development. MethodsBeWo cells were used as surrogates for cytotrophoblasts. The effect of ATRA on ferroptosis sensitivity was assessed on BeWo cells pretreated with ATRA or dimethyl sulfoxide (DMSO; control), following which the LDH-releasing assay was performed. The effects of ATRA pretreatment on the antioxidant defense system (including glutathione [GSH], mitochondrial membrane potential, and heme oxygenase-1 [HMOX1]) in BeWo cells were assessed using assay kits, RT-qPCR, and HMOX1 immunostaining. To evaluate the effect of ATRA on BeWo cells, HMOX1 was silenced in BeWo cells using shRNA. ResultsATRA pretreatment increased ferroptosis resistance in BeWo cells. Although with pretreatment, qPCR indicated upregulation of HMOX1, no significant change was observed in the GSH levels or mitochondrial membrane potential. This was corroborated by intensified immunostaining for heme oxygenase-1 protein (HO-1). Notably, the protective effect of ATRA against ferroptosis was negated when HO-1 was inhibited. Although HMOX1-silenced BeWo cells exhibited heightened ferroptosis sensitivity compared with controls, ATRA pretreatment counteracted ferroptosis in these cells. DiscussionATRA pretreatment promotes BeWo cell viability by suppressing ferroptosis and upregulating HMOX1 and this can be used as a potential therapeutic strategy for addressing placental complications associated with ferroptosis.

Evaluation of the diagnostic utility of NCOA3, Maspin and VHL protein expression in pancreatic ductal adenocarcinoma: An immunohistochemical study

Pancreatic ductal adenocarcinoma (PDAC) is a lethal tumor with a high mortality rate. The distinction between PDAC and chronic pancreatitis is sometimes challenging on routine histopathological examination, highlighting the need to identify biomarkers that can facilitate this distinction. This retrospective study was conducted to evaluate the diagnostic utility of nuclear receptor co-activator 3 (NCOA3), Maspin and Von Hippel-Lindau protein (VHL) immunostaining in PDAC. Eighty cases of PDAC, 46 cases of chronic pancreatitis and 53 normal pancreatic tissue were immunohistochemically assessed using NCOA3, Maspin and VHL antibodies on sections from a tissue microarray. NCOA3, Maspin and VHL were positive in 90 %, 100 % and 35 %, of PDAC cases respectively, whereas NCOA3, Maspin and VHL expressions were positive in 3.8 %, 0 and 100 % of normal pancreatic tissue and in 15.2 %, 21.7 % and 97.8 % of chronic pancreatitis cases respectively. Significant differences were observed between PDAC and other groups regarding NCOA3, Maspin and VHL expression (p < 0.001). The H scores of NCOA3, Maspin and VHL could significantly distinguish between PDAC and normal cases with high sensitivity (90 %, 100 % and 98.75 % respectively) and specificity (100 %, 100 % and 96.23 % respectively). Similar findings were found in the distinction between PDAC and chronic pancreatitis (Sensitivity: 90 %, 95.25 % and 98.75 %; specificity: 100 %, 100 % and 93.48 % for NCOA3, Maspin and VHL respectively). In conclusion, NCOA3 and Maspin were found to be significantly expressed in PDAC compared to non-tumorous tissue while VHL was significantly expressed in non-tumorous tissue. A panel of NCOA3, Maspin and VHL could potentially distinguish PDAC from non-tumorous pancreatic tissue.

Improving corneal permeability of dexamethasone using penetration enhancing agents: First step towards achieving topical drug delivery to the retina

With an ever-increasing burden of vision loss caused by diseases of the posterior ocular segment, there is an unmet clinical need for non-invasive treatment strategies. Topical drug application using eye drops suffers from low to negligible bioavailability to the posterior segment as a result of static and dynamic defensive ocular barriers to penetration, while invasive delivery systems are expensive to administer and suffer potentially severe complications. As the cornea is the main anatomical barrier to uptake of topically applied drugs from the ocular surface, we present an approach to increase corneal permeability of a corticosteroid, dexamethasone sodium-phosphate (DSP), using a novel penetration enhancing agent (PEA). We synthesised a novel polyacetylene (pAc) polymer and compared its activity to two previously described cell penetrating peptide (CPP) based PEAs, TAT and penetratin, with respect to increasing transcorneal permeability of DSP in a rapid ex-vivo porcine corneal assay over 60 min. The transcorneal apparent permeability coefficients (Papp) for diffusion of pAc, and fluorescein isothiocyanate (FITC) conjugated TAT and penetratin were up to 5 times higher (p < 0.001), when compared to controls. When pAc was used in formulation with DSP, an almost 5-fold significant increase was observed in Papp of DSP across the cornea (p = 0.0130), a significant 6-fold increase with TAT (p = 0.0377), and almost 7-fold mean increase with penetratin (p = 0.9540). Furthermore, we investigated whether the PEAs caused any irreversible damage to the barrier integrity of the corneal epithelium by measuring transepithelial electrical resistance (TEER) and immunostaining of tight junction proteins using zonula occludens-1 (ZO-1) and occludin antibodies. There was no damage or structural toxicity, and the barrier integrity was preserved after PEA application. Finally, an in-vitro cytotoxicity assessment of all PEAs in human retinal pigment epithelium cells (ARPE-19) demonstrated that all PEAs were very well-tolerated, with IC50 values of 64.79 mM for pAc and 1335.45 µM and 87.26 µM for TAT and penetratin, respectively. Our results suggest that this drug delivery technology could potentially be used to achieve a significantly higher intraocular therapeutic bioavailability after topical eye drop administration, than currently afforded.

P19 Defining the mechanism of skin barrier restoration by Lactobacillus rhamnosus

Abstract Introduction and aims We aim to investigate the lysate of Lactobacillus rhamnosus GG (LGG), a bacterium previously established as a gut probiotic, and to examine its postulated ability to enhance and restore human skin barrier function. Compromised skin barriers are implicated in various skin conditions including atopic dermatitis. Furthermore, skin barrier function decreases with age leading to dryness and sensitivity. Previous research has shown promising effects of LGG lysates on keratinocytes, including increasing transepidermal electrical resistance, increasing tight junction protein expression, cell migration promotion and mitigation of Staphylococcus aureus toxicity. This study aims to take a closer look at the molecular mechanisms behind the effect of LGG lysates on the skin, including terminal differentiation, lamin dispersal and nuclear degradation in keratinocytes. Methods Normal human epithelial keratinocytes and rat epithelial keratinocytes were grown and treated with the LGG lysate. Western blot analysis was used to determine the relative concentrations of tight junction proteins and other differentiation markers. A phosphoproteomic study was performed to identify key pathways activated by the LGG lysate. A Staphylococcus epidermidis lysate control was used to filter out any nonspecific bacterial effects. Immunostaining of nuclear proteins was used to visualize the effects of LGG lysate on lamin dispersal and nuclear degradation. short hairpin RNA filaggrin knockdown normal human keratinocytes were developed to create a ‘barrier impaired’ model to assess ability of the lysate to ‘rescue’ the skin barrier. Results The LGG lysate was shown to increase tight junction protein expression in normal human keratinocytes and increase keratinocyte terminal differentiation markers. We present Results that show decreased tight junction protein expression in filaggrin knockdown cells. Conclusions These Results show the potential for LGG lysate to enhance and restore skin barrier function and begin to suggest potential mechanisms for this effect.

Moxidectin versus Ivermectin in the prevention and treatment of acute and chronic experimental trichinellosis

The limited activity of the traditional medications against T. spiralis encysted larvae handicaps complete cure of trichinellosis till now due to decreased permeability and absorption through tissues. MOX is listed worldwide for prevention and treatment of several internal and external nematodes. Consequently, the aim of this work was to investigate the effect of moxidectin versus ivermectin on experimental acute and chronic trichinellosis and to illuminate the potential mechanisms of their effects. 105 Mice were divided into four groups; Group I: Uninfected healthy control; Group II: Infected untreated control; Group III: Infected and treated with IVM and Group IV: Infected and treated with MOX. The groups (II, III and IV) were later subdivided equally into three subgroups (a, b, and c) according to the stage of treatment. Parasitological counting of adults and larvae besides immune-histopathological examination of intestines and muscles were done. Results exhibited that both IVM and MOX succeeded in reducing adults and larvae counts with higher potential of MOX in both intestinal and muscle phase. The preeminence of MOX was indicated by decreased inflammation, a significant reduction in the microvascular density (CD31 immunostaining) as well as a reduction in the percentage of fibroblast activation protein (FAP) immunostaining in muscle tissues. Accordingly, the current work recommends moxidectin as an innovative treatment for trichinellosis.