Immunohistochemical Assessment Research Articles

(005) Immunohistochemical Staining of the 12:00 Vestibule in NPV Patients: First Ever Assessment

Abstract Introduction In 2004, Bornstein and colleagues performed immunohistochemical (IHC) staining of excised vestibular specimens in patients with vestibular pain. They demonstrated that these specimens contained > 8 CD117 immunopositive stained cells per high-power field (HPF), consistent with mast cells, and excess S100 stained immunopositive cells, consistent with nerves-far more compared to control tissue. However, Bornstein et al. only studied the 1:00-11:00 vestibule in this condition, now referred to as neuroproliferative vestibulodynia (NPV). To the best of our knowledge, the 12:00 region of the vestibule, below the clitoral frenulae and above the urethral meatus, has not been examined with IHC staining. Pain in the 12:00 region can be particularly bothersome for patients and poses several challenges for successful treatment. Objective To report IHC staining findings in the excised 12:00 vestibule specimen and associated clinical characteristics of NPV patients. Methods Retrospective chart review performed. Slides of IHC-stained vestibular tissue specimens from the 12:00 vestibule were examined and high-resolution digital microscopy images were captured under 100x and 200x magnification. A least 2 photomicrographs at both magnifications were taken for each slide that included representative areas of epithelial basement membrane and the adjacent subepithelial regions. Fractional area of positive immunostaining in all photomicrographs was assessed and mean values calculated using computer-assisted histometry by ImageJ. Results A total of 50 patients had complete vestibulectomy including 12:00 and 3 had regional vestibulectomy for only the 12:00 area. Using manual counting as applied by Bornstein et al., 53 excised 12:00 specimens stained for CD117 had a median immunopositive cell count, consistent with mast cells, of 29.5, greater than the described 8 mast cells per HPF. Using computer-assisted histometry, tissue stained for CD117 had a median fractional area of 0.73 in the 12:00 region. Tissues stained for PGP9.5 had a median fractional area of 0.31 in the 12:00 region. Overall, there was no difference between lifelong NPV (LNPV) and acquired NPV (ANPV) patients in the cell count or median fractional area for CD117-immunopositive or PGP9.5-immunopositive staining in the 12:00 region. In LNPV patients who had excision of both the 1:00-11:00 and 12:00 regions, the 12:00 region had significantly less PGP9.5 immunopositive fractional area than the 1:00-11:00 region of the vestibule (p = 0.001). Their H&E pathology findings, including severity grading of the subepithelial inflammatory infiltrate, did not predict the IHC assessment of either CD117 or PGP9.5 in the 12:00 region. The median cotton-tipped swab test pain score was 5 out of 10 for the 12:00 region vs 7 for the rest of the vestibule. Conclusions Current surgical practices often do not include the 12:00 region, likely due to lack of data demonstrating excess mast cells and nerves in this specific region. Our results suggest that there are, in fact, a high density of immunopositive histochemical stains consistent with mast cells and nerves in the 12:00 region and high reported pain scores in both 12:00 and 1:00-11:00 vestibule. Thus, NPV surgical treatments should include, when appropriate, the 12:00 vestibule, as NPV is a field disease involving the entire vestibule. Disclosure No.

D-limonene inhibits peritoneal adhesion formation in rats via anti-inflammatory, anti-angiogenic, and antioxidative effects.

The aim of this research was to investigate the effects of D-limonene on decreasing post-operative adhesion in rats and to understand the mechanisms involved. Peritoneal adhesions were induced by creating different incisions and excising a 1 × 1cm section of the peritoneum. The experimental groups included a sham group, a control group in which peritoneal adhesions were induced without any treatment, and two treatment groups in which animals received D-limonene with dosages of 25 and 50mg/kg after inducing peritoneal adhesions. Macroscopic examination of adhesions showed that both treatment groups had reduced adhesion bands in comparison to the control group. Immunohistochemical assessment of TGF-β1, TNF-α, and VEGF on day 14 revealed a significant increment in the level of immunopositive cells for the mentioned markers in the control group, whereas administration of limonene in both doses significantly reduced levels of TGF-β1, TNF-α, and VEGF (P < 0.05). Induction of peritoneal adhesions in the control group significantly increased TGF-β1, TNF-α, and VEGF on days 3 and 14 in western blot evaluation, while treatment with limonene significantly reduced TNF-α level on day 14 (P < 0.05). Moreover, VEGF levels in both treatment groups significantly reduced on days 3 and 14. In the control group, a significant increment in the levels of MDA and NO and a notable decline in the levels of GPX, CAT was observed (P < 0.05). Limonene 50 group significantly reduced MDA level and increased GPx and CAT levels on day 14 (P < 0.05). In summary, D-limonene reduced adhesion bands, inflammatory cytokines, angiogenesis, and oxidative stress.

Prompt Placental Histopathological and Immunohistochemical Assessment after SARS-CoV-2 Infection during Pregnancy-Our Perspective of a Small Group.

Research indicates compelling evidence of SARS-CoV-2 vertical transmission as a result of placental pathology. This study offers an approach to histopathological and immunohistochemical placental observations from SARS-CoV-2-positive mothers compared to negative ones. Out of the 44 examined placentas, 24 were collected from patients with a SARS-CoV-2 infection during pregnancy and 20 were collected from patients without infection. The disease group showed strong SARS-CoV-2 positivity of the membranes, trophoblasts, and fetal villous macrophages. Most infections occurred during the third trimester of pregnancy (66.6%). Pathology revealed areas consistent with avascular villi (AV) and thrombi in the chorionic vessels and umbilical cord in the positive group, suggesting fetal vascular malperfusion (FVM). This study shows SARS-CoV-2 has an impact on coagulation, demonstrated by fetal thrombotic vasculopathy (p = 0.01) and fibrin deposition (p = 0.01). Other observed features included infarction (17%), perivillous fibrin deposition (29%), intervillous fibrin (25%), delayed placental maturation (8.3%), chorangiosis (13%), chorioamnionitis (8.3%), and meconium (21%). The negative control group revealed only one case of placental infarction (5%), intervillous fibrin (5%), delayed placental maturation (5%), and chorioamnionitis (5%) and two cases of meconium (19%). Our study sheds light on the changes and differences that occurred in placentas from SARS-CoV-2-infected mothers and the control group. Further research is necessary to definitively establish whether SARS-CoV-2 is the primary culprit behind these intricate complications.

Human Umbilical Cord Mesenchymal Stem Cells Alleviate Rat Knee Osteoarthritis via Activating Wnt/ β-catenin Signaling Pathway.

Osteoarthritis (OA) is a chronic disease characterized by joint cartilage degeneration, destruction, and osteogenic hyperplasia. Human umbilical cord mesenchymal stem cells (hUCMSCs) have attracted increasing research interest due to their high clonogenic, proliferative, and migratory potential, as well as their improved secretion of relevant chondrogenic factors. This study evaluated the therapeutic potential and underlying mechanism of hUC-MSCs in alleviating pathological symptoms of OA. For the in vivo study, OA rats were established by the Hulth method to observe the therapeutic effect of intra-articular injection of hUC-MSCs. X-ray tests, gross observations, and histological and immunohistochemical assessments were conducted in rats. Levels of interleukin-1 beta (IL-1β), IL-6, matrix metalloproteinase-13 (MMP-13), and tissue inhibitor matrix metalloproteinase-1 in rats' synovial fluid were measured using enzyme-linked immunosorbent assay kits. For the in vitro study, hUC-MSCs and chondrocytes were cultured to explore the effect and underlying mechanisms of hUC-MSCs on OA. Apoptosis, proliferation, and glycosaminoglycan (GAG) were measured in the chondrocytes. The relative expression of aggrecan, COL-2, and SOX-9 mRNA was quantified by real-time polymerase chain reaction. Expressions of Wnt/β-catenin signaling molecules were measured by Western blot. We found that intra-articular injection of hUC-MSCs reduced the combined score, increased the expression of collagen II, and decreased the expression of MMP-13, IL-1β, and IL-6 in rat knee joints. Additionally, hUC-MSCs increased the content of GAGs, inhibited chondrocyte apoptosis, and promoted chondrocyte proliferation. The expression of aggrecan, COL-2, and SOX-9 mRNA in chondrocytes was promoted by hUC-MSCs via activation of the Wnt/β-catenin signaling pathway. Overall, this study demonstrated that hUC-MSCs induce the secretion of some cytokines via the paracrine function to activate the Wnt/β-catenin signaling pathway to reduce the pathological condition of OA and maintain the proper expression of cytokines and extracellular matrix proteins.

Abstract B016: Recapitulation of gene expression-based immune subtypes of breast cancer using immunohistochemical staining

Abstract Using gene expression profiling data, we previously identified three immune subtypes (low-TIL, high-TIL, high-ISG) of breast cancer (BC) among Chinese BC patients in Hong Kong (HK) (Zhu et al., Breast Cancer Res, 2017). To investigate if standard in situ immunohistochemical (IHC) staining could capture the BC immune landscape, we analyzed archival tissue sections from 148 HKBC patients who were previously characterized based on gene expression. Dual IHC staining for 8 immune markers (CD3, CD8, CD20, FOXP3, CD68, CD163, PDL1, and IDO1) and PanCK was performed. Digitized whole slide images for each marker were analyzed using high-accuracy machine learning algorithms trained to quantify the percentage of positively stained regions. Strong correlations were found between IHC immune markers and gene expression-based immune and intrinsic subtypes. Compared to patients with lower level of tumor-infiltrating lymphocytes (low-TIL) classified based on gene expression data, those in the high-TIL subtype (characterized by high T cell and immune checkpoint gene expression) showed higher proportions of CD3, CD8, CD20, FOXP3, CD68, and PDL1 IHC positively stained cells. Similarly, the high-ISG subtype (with increased interferon-stimulated gene expression and TP53 somatic mutations) had higher proportions of CD3, CD8, CD20, FOXP3, and CD68 stained cells than the low-TIL subtype and in particular exhibited high CD163 staining, indicative of a proinflammatory tumor microenvironment. CD8, CD20, CD163, PDL1, and IDO1 expression varied significantly by PAM50 subtypes, with luminal (A and B) subtypes showing lower staining than non-luminal (HER2 and Basal) subtypes. A distinctive feature, however, of luminal B tumors was the higher infiltration by CD163-expressing cells when compared with the luminal A subtype. Except for FOXP3, all examined IHC markers showed significantly higher expression in patients with somatic TP53 mutations (CD3: P= 0.003, CD8: P= 0.005, CD20: P= 0.003, CD68: P= 0.002, CD163: P= 0.002, PDL1: P= 0.003, and IDO1: P= 0.013). On the other hand, expression of these markers did not significantly vary by PIK3CA mutation or germline APOBEC3A/B deletion polymorphism, which is more prevalent among East Asians than other populations and was previously linked to BC risk and immune environment. Of the BC risk factors assessed, only body mass index (BMI) demonstrated associations with immune cell densities, with higher BMI associated with lower expression of CD3, CD8, and CD20. This study sheds further light on the in situ immune landscape of gene expression-based BC subtypes, suggesting that standard IHC assessment of immune markers could refine BC subtype classification for clinical and epidemiological use. Citation Format: Mustapha Abubakar, Hela Koka, Priscilla Lee, Scott Lawrence, Karun Mutreja, Difei Wang, Bin Zhu, Shelly Tse, Xiaohong R Yang. Recapitulation of gene expression-based immune subtypes of breast cancer using immunohistochemical staining [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B016.

Polydioxanone Enhances Bone Regeneration After Resection and Reconstruction of Rat Femur with rhBMP2.

The aim of this study was to assess the bone regeneration potential of a polydioxanone (PDO) scaffold together with recombinant human bone morphogenetic protein-2 (rhBMP-2) for the reconstruction of large bone defect. In total, 24 male rats (6 months old) were subjected to bilateral femoral stabilization using titanium plates to create a 2 mm gap, and reconstruction using rhBMP-2 (Infuse®; 3.25 μg). The bone defects were covered with PDO (PDO group), or with titanium mesh (Ti group). Animals were euthanized on days 14 and 60. Simultaneously, 16 rats received PDO and Ti in their dorsum for the purpose of biocompatibility analysis at 3, 5, 7, and 10 days postoperatively. X-ray densitometry showed a higher density in the PDO group on day 14. On day 60, coverage of the bone defect with PDO showed a larger quantity of newly formed bone than that found for the Ti group, a lower inflammatory infiltrate value, and a more significant number of blood vessels on day 14. By immunohistochemical assessment, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) showed higher labeling on day 14 in the PDO group. On day 60, bone morphogenetic protein-2 (BMP-2) showed higher labeling in the PDO group, whereas Ti showed higher labeling for osteoprotegerin, nuclear factor kappa B ligand-activating receptor, RUNX2, and OCN. Furthermore, biocompatibility analysis showed a higher inflammatory response in the Ti group. The PDO scaffold enhanced bone regeneration when associated with rhBMP-2 in rat femur reconstruction. Impact statement Regeneration of segmental bone defects is a difficult task, and several techniques and materials have been used. Recent advances in the production of synthetic polymers, such as polydioxanone (PDO), produced by three-dimensional printing, have shown distinct characteristics that could improve tissue regeneration even in an important bone defect. The present preclinical study showed that PDO membranes used as scaffolds to carry recombinant human bone morphogenetic protein-2 (rhBMP-2) improved bone tissue regeneration by more than 8-fold when compared with titanium mesh, suggesting that PDO membranes could be a feasible and useful material for use in guided bone regeneration. (In English, viable is only used for living creatures capable of sustaining life.

Citrus clementine Peel Essential Oil Ameliorates Potassium Dichromate-Induced Lung Injury: Insights into the PI3K/AKT Pathway.

Acute Lung Injury (ALI) is a life-threatening syndrome that has been identified as a potential complication of COVID-19. There is a critical need to shed light on the underlying mechanistic pathways and explore novel therapeutic strategies. This study aimed to examine the potential therapeutic effects of Citrus clementine essential oil (CCEO) in treating potassium dichromate (PDC)-induced ALI. The chemical profile of CCEO was created through GC-MS analysis. An in vivo study in rats was conducted to evaluate the effect of CCEO administrated via two different delivery systems (oral/inhalation) in mitigating acute lung injury (ALI) induced by intranasal instillation of PDC. Eight volatile compounds were identified, with monoterpene hydrocarbons accounting for 97.03% of the identified constituents, including 88.84% of D-limonene. CCEO at doses of 100 and 200 mg/kg bw exhibited antioxidant and anti-inflammatory properties. These significant antioxidant properties were revealed through the reduction of malondialdehyde (MDA) and the restoration of reduced glutathione (GSH). In addition, inflammation reduction was observed by decreasing levels of cytokines tumor necrosis factor-α and tumor growth factor-β (TNF-α and TGF-β), along with an increase in phosphatidylinositide-3-kinase (PI3K) and Akt overexpression in lung tissue homogenate, in both oral and inhalation routes, compared to the PDC-induced group. These results were supported by histopathological studies and immunohistochemical assessment of TGF-β levels in lung tissues. These findings revealed that CCEO plays an integral role in relieving ALI induced by intranasal PDC and suggests it as a promising remedy.

Fracture-Induced Effects on the Onset &amp; Progression of Alzheimer’s Disease

Alzheimer’s disease and related dementias (AD/ADRD) are multifactorial, highly heterogeneous, and complex age-dependent disorders that severely affect memory and cognitive function, impacting nearly 35.6 million people worldwide. In the elderly, dementia increases the risk of falls and fractures by 2-3 times, due in part to neurovascular instability, low bone mineral density due to pre-existing osteoporosis, and poor musculature supporting joints due to cachexia and/or sarcopenia.&#x0D; While the occurrence of fractures due to AD/ADRD is well documented, an association between fractures and AD/ADRD onset or progression is underappreciated and warrants additional investigation. We aim to investigate the mechanistic actions underlying fracture healing as a precipitating event for AD/ADRD pathogenesis.&#x0D; Four-month-old, male, 5xFAD (AD model) and wild-type control (C57BL/6) mice were divided into 2 groups: surgically induced femoral fractures and uninjured mice. Prior to surgery mice underwent baseline AD behavior testing including: spontaneous alternation in the y-maze, light-dark exploration in the open field, and active place avoidance assays. Mice are undergoing weekly x-ray imaging to monitor fracture healing progression and longitudinal AD behavior testing. 22 weeks post-surgery mice will be euthanized, and femurs and brains collected. Femurs will undergo uCT imaging and histological assessment of bone healing and immunohistochemical assessment of inflammatory markers. Brains will be processed for histology and neuroinflammatory marker analysis, including Aβ plaque deposition, tau tangles, neuronal survival, neurogenesis, and activation/proliferation of microglia and astrocytes.&#x0D; At the conclusion of the study, we expect to see an increase in neuroinflammatory markers and delayed fracture healing in the experimental 5xFAD group. We anticipate that compared to uninjured controls, femoral fracture results in cognitive decline, Aβaccumulation/neurodegeneration, increases in neuroinflammation, and vascular impairment. We anticipate finding a correlation between fracture and worsened AD outcomes. By uncovering the mechanisms underlying this relationship, we hope to guide future studies to develop more robust therapeutics.

The Combined Intraosseous Administration of Orthobiologics Outperformed Isolated Intra-articular Injections in Alleviating Pain and Cartilage Degeneration in a Rat Model of MIA-Induced Knee Osteoarthritis

Background: Intra-articular (IA) platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMAC) injections have shown efficacy and safety in treating osteoarthritis (OA). However, the effectiveness and mechanisms of combined intraosseous (IO) administration of these orthobiologics have yet to be explored. Purpose/Hypothesis: The purpose of this study was to evaluate the effect on pain, cartilage, synovium/infrapatellar fat pad (IFP), and subchondral bone in rat knee OA, comparing isolated IA with combined IA and IO (IA+IO) injections of PRP or BMAC. It was hypothesized that combined injections would be superior to sole IA injections. Study Design: Controlled laboratory study. Methods: A total of 48 rats were divided into 6 groups: sham (only joint puncture during OA induction with IA+IO saline injection treatment) and 5 groups with OA induction, control (IA+IO saline injection), PRP (IA PRP+IO saline injection), BMAC IA (IA BMAC+IO saline injection), PRP IA+IO (IA+IO PRP injection), and BMAC IA+IO (IA+IO BMAC injection). OA was induced by IA injection of monosodium iodoacetate (MIA). Rats were administered different orthobiologics according to their grouping 3 weeks after the MIA injection. Pain changes were evaluated using the weightbearing ratio assay at weeks 3, 4, 5, 7, and 9 after OA induction. Rats were euthanized at week 9 for gross, radiological, histological, immunohistochemical, and immunofluorescence assessments of cartilage, synovium, and subchondral bone. Results: Compared with the control group, all orthobiologics injection groups had reduced joint pain. Compared with IA injection, IA+IO injections provided superior pain relief by suppressing calcitonin gene-related peptide and substance P in both the synovium/IFP and subchondral bone. IA+IO injections slowed the progression of subchondral bone lesions by inhibiting CD31hiEmcnhi vessel formation and excessive osteoclast and osteoblast turnover while preserving subchondral bone microarchitecture, slowing cartilage degeneration. However, IA+IO injections did not outperform isolated IA injections in reducing synovitis and synovium/IFP fibrosis. Compared with PRP, BMAC exhibited superior inhibition of pain-related mediators, but no significant differences were observed in synovitis suppression, infrapatellar fat pad fibrosis, and subchondral bone protection. Conclusion: IA+IO injections of orthobiologics were more effective in relieving pain, slowing cartilage degeneration, and inhibiting abnormal vascularization and remodeling compared with isolated IA injections. BMAC showed superior pain relief in the synovium/IFP and subchondral bone compared with PRP. Further research is needed to optimize PRP and BMAC components for enhanced efficacy in OA management. Clinical Relevance: Our findings contribute to advancing the understanding of pain relief mechanisms and support the endorsement of IO injection of orthobiologics for the treatment of OA and joint pain.

Glaucoma, More than Meets the Eye: Patterns of Demyelination Revealed in Human Postmortem Glaucomatous Optic Nerve.

Glaucoma is a neurodegenerative disease affecting millions worldwide, characterised by retinal ganglion cell (RGC) degeneration which leads to blindness in more advanced cases. Although the pathogenesis and underlying mechanisms of glaucoma are not fully understood, there are theories that hint at demyelination playing a role in the disease process. Demyelination, or the degeneration of the myelin sheath surrounding axons, has been found in previous studies using animal models of glaucoma and clinical assessments of glaucoma patients. However, this has not been fully realised or quantified in glaucoma patients. Utilising postmortem optic nerve samples from glaucoma and healthy subjects, various immunohistochemical and morphological assessments were performed to determine the extent, if any, of demyelination in glaucomatous optic nerves. Our findings revealed that alongside nerve shrinkage and degeneration of nerve tissue fascicles, there were significantly less myelin proteins, specifically myelin basic protein (MBP), in glaucoma optic nerves. Additionally, the loss of MBP was correlated with decreased oligodendrocyte (OLG) precursors and increasing glial activity. This further supports previous evidence that demyelination may be a secondary degenerative process associated with glaucoma disease progression. Not only do these results provide evidence for potential disease mechanisms, but this is also the first study to quantify optic nerve demyelination in glaucoma postmortem tissue.

Fetal progenitor cells for treatment of chronic limb ischemia.

This study investigated the therapeutic potential of fetal progenitor cells (FPCs) in the treatment of chronic non-healing wounds and ulcers associated with chronic limb ischemia (CLI). The research aimed to elucidate the mechanism of action of FPCs and evaluate their efficacy and safety in CLI patients. The researchers isolated FPCs from aborted human fetal liver, brain, and skin tissues and thoroughly characterized them. The preclinical phase of the study involved assessing the effects of FPCs in a rat model of CLI. Subsequently, a randomized controlled clinical trial was conducted to compare the efficacy of FPCs with standard treatment and autologous bone marrow mononuclear cells in CLI patients. The clinical trial lasted 12 months, with a follow-up period of 24-36 months. The primary outcomes included wound healing, frequency of major and minor amputations, pain reduction, and the incidence of complications. Secondary outcomes involved changes in local hemodynamics and histological, ultrastructural, and immunohistochemical assessments of angiogenesis. In the animal model, FPC treatment significantly enhanced angiogenesis and accelerated healing of ischemic wounds compared to controls. The clinical trial in CLI patients demonstrated that the FPC therapy achieved substantially higher rates of complete wound closure, prevention of major amputation, pain reduction, and improvement in ankle-brachial index compared to control groups. Notably, the study reported no serious adverse events. FPC therapy exhibited remarkable efficacy in promoting the healing of ischemic wounds, preventing amputation, and improving symptoms and quality of life in patients with CLI. The proangiogenic and provasculogenic effects of FPCs may be attributed to their ability to secrete specific growth factors. These findings provide new insights into the development of cellular therapeutic angiogenesis as a promising approach for the treatment of peripheral arterial diseases.

Neural tissue tolerance to synthetic dural mater graft implantation in a rabbit durotomy model.

In neurosurgical interventions, effective closure of the dura mater is essential to prevent cerebrospinal fluid leakage and minimize post-operative complications. Biodegradable synthetic materials have the potential to be used as dura mater grafts owing to their regenerative properties and low immunogenicity. This study evaluated the safety of ArtiFascia, a synthetic dura mater graft composed of poly(l-lactic-co-caprolactone acid) and poly(d-lactic-co-caprolactone acid), in a rabbit durotomy model. Previously, ArtiFascia demonstrated positive local tolerance and biodegradability in a 12-month preclinical trial. Here, specialized stains were used to evaluate potential brain damage associated with ArtiFascia use. Histochemical and immunohistochemical assessments included Luxol Fast Blue, cresyl Violet, Masson's Trichrome, neuronal nuclei,, Glial Fibrillary Acidic Protein, and ionized calcium-binding adaptor molecule 1 stains. The stained slides were graded based on the brain-specific reactions. The results showed no damage to the underlying brain tissue for either the ArtiFascia or control implants. Neither inflammation nor neuronal loss was evident, corroborating the safety of the ArtiFascia. This approach, combined with previous histopathological analyses, strengthens the safety profile of ArtiFascia and sets a benchmark for biodegradable material assessment in dura graft applications. This study aligns with the Food and Drug Administration guidelines and offers a comprehensive evaluation of the potential neural tissue effects of synthetic dura mater grafts.

Uterine tumor resembling ovarian sex-cord tumor - a rare gynecological neoplasm.

Uterine tumor resembling ovarian sex-cord tumor is a rare group of uterine neoplasms with unknown histogenesis and differentiation towards ovarian sex-cord elements. They are benign in nature with low malignancy potential. Diagnosis is based on immunohistochemistry and morphological features, and the distinction from other more malignant differentials is paramount to correctly individualizing treatment.

CXCL10 Expression in Human Colorectal Cancer Tissue and its Correlation With Serum Levels of CXCL10.

CXCL10, a member of the CXC chemokine family, plays a crucial role in immune response by facilitating the chemotaxis of CXCR3-positive immune cells. We examined the expression of CXCL10 to unravel its functional significance in colorectal cancer. Bioinformatics analysis was performed to investigate CXCL10 expression and its clinicopathological relevance. Subsequently, we examined the correlation between the serum levels of CXCL10 and its expression within cancer tissues. Analysis of the TCGA database revealed that elevated CXCL10 expression in CRC tissues correlates with improved long-term survival and is inversely associated with lymph node infiltration and metastasis. Insights from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes further established a connection between increased CXCL10 and co-regulated gene expression with enhanced immune activation and regulation, mediated by the inhibition of the NOD-like receptor signaling pathway. Single-cell analysis pinpointed myeloid cells and macrophages as the primary sources of CXCL10. Immunohistochemical assessments revealed that a subset of cancer cells and macrophages are positive for CXCL10 expression. CXCL10-positive cells are predominantly located at the invasive front of the tumor. Intriguingly, our findings reveal an inverse correlation between serum CXCL10 levels and its expression in cancer tissues. The expression of CXCL10 may play a role in mediating the inflammatory responses at the invasive front in colorectal cancer and is observed to be inversely correlated with serum CXCL10 levels. It is pivotal to elucidate the distinct roles of CXCL10 in colorectal cancer, particularly different functions of cancer-tissue CXCL10 from serum CXCL10.

Co expression of EGFR and CD10 in patients with phyllodes tumors of the breast: a single center experience in North Western Algeria.

Breast phyllodes tumors (BPT) have variable malignant potential, their histological classification remains insufficient for an accurate diagnosis. We attempted to investigate CD10 (Cluster of differentiation 10) and EGFR (Epidermal growth factor receptor) expression in BPT in order to highlight their diagnostic and prognostic values. Eight patients with BPT are recruited from January 2014 to December 2020 and immunohistochemical assessment of CD10 and EGFR is realized. Median age was 27±15.2, the mean tumor size was 9.63±10.21. Only malignant tumours showed expression for EGFR. Borderline and malignant tumors were CD10 positive. Patients overexpressing CD10 were postmenopausal with great tumor size, 25% of these were sarcomatous. Coexistence of CD10 and EGFR overexpression was found in 25% of cases and was associated with age (P=0.008), tumor size (P=0.030) and hitologic types (P=0.014). PC1 and PC2, were extracted, they accounted cumulatively for 94.7% of the variance of the data analysed, it suggests that patient's age and histological type of tumor have significant association with CD10 and EGFR expression in BPT. EGFR and CD10 overexpressed combined proteins in phyllode tumors constitute, with histopathological parameters, an important prognostic factor as well as a promising potential targets.

Pathological and immunohistochemical assessment of the impact of three different strains of swine enteric coronaviruses in the intestinal barrier

Swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV) or transmissible gastroenteritis virus (TGEV), have risen concern for the porcine industry and research community due to the increase in their virulence, their potential recombination capacity and the emergence of new variants. This in vivo study aims to compare the impact of three different strains of swine enteric coronaviruses [(two G1b (S-INDEL) PEDV strains and a recombinant TGEV-PEDV or Swine enteric coronavirus (SeCoV)] in the intestine of 3-weeks-old infected piglets, focusing on the pathology and main components of the intestinal barrier, including the number of goblet cells, and the expression of IgA as well as FoxP3, a regulatory T cell marker. Severity of lesions was evidenced in the three infected groups and was highly correlated with the viral load in feces and the frequency of viral antigen-positive cells. Furthermore, higher cellular death together with an increase in the expression of the FoxP3 marker was detected in the duodenum and jejunum of infected animals at 3 days post-infection. Our results highlight a recruitment of FoxP3+ cells in the small intestine of infected animals which may represent a response to the tissue damage caused by viral replication and cell death. Further studies should be addressed to determine the potential role of these cells during swine enteric coronavirus infections.

Immunohistochemical and behavioral assessment of FKBP51‐Hsp90 interaction‐deficient mouse crossed with Alzheimer tau transgenic Tg4510 mice

AbstractBackgroundFK506‐binding protein 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) associated co‐chaperone which has recently emerged as a possible Alzheimer disease (AD) therapeutic target involved in tau metabolism. Increased expression of FKBP51 was found in brains of aged mice and AD patients and overexpression of FKBP51 in mice impairs cognitive function in vivo. To elucidate the role of FKBP51‐Hsp90 complex in tau biology in vivo we have crossed well characterized tau transgenic Tg4510 mice with our novel FKBP51‐Hsp90 interaction‐deficient mice (Fkbp5TPRmut).Methods• Using PCR amplification, we have confirmed correct genotype of crossings.• Immunohistochemical staining of brains of 4 months‐old animals with total tau and phospho‐tau antibodies.• Behavior assessment of cognition and memory using open field, novel object recognition, Y‐maze, and fear conditioning tests.Results• Successful crossings between Fkbp5TPRmut and Tg4510 mice have been obtained• We have confirmed phospho‐tau expression in the cortex and hippocampus of 4 months‐old transgene‐expressing animals (positive controls).• Immunohistochemical assessment of phospho‐tau expression in the cortex and hippocampus of wild‐type and Fkbp5TPRmut x Tg4510 crossings is ongoing.• Behavioral assessment of Wt x Tg4510 and Fkbp5TPRmut x Tg4510 crossings is ongoing.Conclusion• Fkbp5TPRmut crossings with Tg4510 mice do not display harmful phenotype.• Behavioral and immunohistochemical evaluation experiments are ongoing and will soon be completed.

Relationships between CD34-, CD105- and bcl-2-Expression Levels and Contrast-Enhanced Ultrasound-Based Differential Diagnosis of Adnexal Tumours

The relationships between CEUS parameters of adnexal tumours and postoperative immunohistochemical assessments of CD34, CD105 and bcl-2 were analysed. This study aimed to investigate whether contrast-enhanced ultrasonography (CEUS) parameters depend on the microvascular density of the tumour lesion found after surgery. Fifty-one patients with a diagnosis of adnexal tumours were included in this single-centre, prospective study. Participants underwent preoperative CEUS (contrast-enhanced ultrasound). Colour Doppler enhancement characterisation parameters (Ystart, Ymax and S) were determined. Immunohistochemical examination of histological specimens of the adnexal lesions was then carried out to determine the expression levels of the CD34, CD105 and bcl-2 proteins. Relationships between the aforementioned parameters were investigated. No significant statistical correlations were observed between CD34, CD105 and bcl2 expression levels and CEUS parameters, independently of whether the operated lesion was malignant or benign. Transvaginal CEUS is diagnostic for the detection of pathological neoplastic vascularisation of an adnexal lesion independent of the density of microcapillaries found postoperatively.

Immunohistochemical Tumor Expression Predicts Response to CD3xCD20 Bispecific Antibodies in Patients with Relapsed/Refractory B Cell Lymphoma

Introduction Several studies show that CD3xCD20 bispecific antibodies show promising results in relapsed or refractory B-cell lymphomas and may have a milder adverse effect profile compared to chimeric antigen receptor T cell therapy (CAR-T). Tumor loss of antibody target (i.e. CD19 or PD1) have shown to negatively affect treatment outcomes in CD19-CAR-T cell therapy in DLBCL, and anti-PD1 therapy in Hodgkin lymphoma. It has, to our knowledge, not been reported how loss of CD20 expression and other immunohistochemical (IHC) expression patterns affects treatment outcomes in patients treated with CD3xCD20 antibodies. Methods We collected data on all patients in Denmark who received CD3xCD20 bispecific antibodies from 2017 to 2023. Electronic health records were assessed for general baseline characteristics and response according to Lugano criteria. Pathology reports on pre- and post-CD3xCD20 therapy lymphoma samples were collected and assessed on IHC analysis of CD20, Ki67, CD10, Bcl2, Bcl6, MUM1, germinal center B-cell (GCB) subtype and non-germinal center B-cell subtypes (non-GCB) according to Hans algorithm, as well as reports on cytogenetic FISH analysis regarding MYC with BCL2 and/or BCL6 rearrangements. To determine whether an expression was weak, negative, or strong the assessment of the original pathology reports was used. Results Pre-treatment IHC assessments were available in 122 patients. These were distributed across 81 patients with DLBCL, 32 with FL and 9 with other lymphoid neoplasms (MZL, HL, CLL). Of these, 109 had a strong CD20 expression, 10 had a weak CD20 expression, while 3 had no expression of CD20. Patients with a weak CD20 expression before treatment had an overall survival (OS) rate after 12 months at 40% (95% CI, 9.5-70) compared to 69% in patients with strong CD20 expression (95% CI, 45-61) (Figure 1, p=0.08). In a univariable Cox regression, we found that negative CD20 expression and higher Ki67 was significantly associated with inferior OS (Table 1, respectively HR=2.11 and HR=1.02 (per %-change in Ki67)). Worse performance status and a higher number of previous lines of treatment were also significantly associated with inferior overall survival (respectively HR=1.85 and HR=1.20). Surprisingly, BCL6 rearrangement was significantly associated with superior OS (HR=0.46). In a multivariable cox regression using OS as the endpoint, weak and negative IHC expression of CD20 was significantly associated with inferior OS (respectively HR=3.29, 95% CI 1.32-8.23, p=0.011 and HR=6.79 95% CI 1.25-36.76, p=0.026). Positive BCL6 compared to negative was associated with increased OS (HR =0.48, 95% CI 0.24 - 0.94, p=0.032). Analyzing only patients with DLBCL (n=81), weak and negative IHC expression of CD20 were the only factors to remain significantly associated with inferior overall survival (respectively HR=3.01, 95% CI 1.10-8.28, p=0.032 and HR=7.26, 95% CI 1.35-43.35, p=0.026). BCL6 rearrangement was associated with an increased overall survival, however only borderline statistically significant (HR=0.53, 95% CI 0.26-1.08, p=0.082). With regards to treatment response weak CD20 expression, worse performance status, higher Ki67 and a higher number of prior lines of treatment was in univariable analysis significantly associated with not being in a complete remission (CR) (respectively HR=2.28, HR=1.73, HR=1.01(per %-change in Ki67), HR=1.23 (per prior line of treatment). In a multivariable cox regression only weak and negative CD20 expression remained significantly associated with not being in CR (respectively HR=2.40, 95% CI 1.1-5.25, p=0.028 and HR=5.7 95% CI 1.14-28.58, p=0.034). Of the 48 patients who had biopsy-confirmed relapse, 29 had a weak or negative IHC CD20 expression (24 with a negative expression and 5 with a weak expression). Of these 29 patients, 24 had a strong pre-treatment IHC CD20 expression. Conclusions To our knowledge, we here present the first long-term analysis of the prognostic effect of tumor immunohistochemistry properties in patients treated with CD3xCD20 bispecific antibodies. We find that CD20 expression before treatment is an important prognostic factor, as patients with lymphomas with weak CD20 expression yield significantly poorer responses and decreased overall survival. Additionally, we find that BCL6 rearrangement is associated with superior overall survival in patients treated with CD3XCD20 bispecific antibodies.

Does Dactyloctenium aegyptium ethanolic extract protect against hormonal, histological and immunohistochemical alterations induced by sodium fluoride in rat adrenal gland?

ABSTRACT This work aimed to perform a biochemical, histological, and immunohistochemical assessment of the influence of sodium fluoride on the adrenal gland of rats and the possible ameliorating role of Dactyloctenium aegyptium (D. aegyptium). Thirty-five male albino rats were divided into five equal groups: group I served as the control group, group II received sodium fluoride (5 mg/kg), group III received D. aegyptium (200 mg/kg), and groups IV and V received sodium fluoride and D. aegyptium (100 and 200 mg/kg respectively) simultaneously for 28 days. Aldosterone, corticosterone, nitric oxide, sodium, potassium, and chloride were measured in serum samples. Adrenal gland specimens were processed for histological, histochemical, and immunohistochemical studies. The sodium fluoride group recorded a significant reduction in serum levels of aldosterone, corticosterone, potassium, and chloride (p ≤ 0.0001) and a significant increase in nitric oxide (p ≤ 0.0001) and serum sodium (p = 0.0076) when compared with the control group. Histological changes in the sodium fluoride treated group showed cytoplasmic vacuolation, scattered apoptotic cells, and hemorrhage. The sodium fluoride treatment significantly decreased glycogen (p = 0.0002), total protein) p = 0.0007), and DNA (p ≤ 0.0001) and increased inducible nitric oxide synthase (p = 0.0001). Co-treated groups with D. aegyptium showed almost average values for most parameters and a nearly standard architecture. The present data answers the question of our research that D. aegyptium ethanolic extract could protect against sodium fluoride’s adverse effects in rat adrenal glands on the physiological and histological levels.