Ki-67 Antibody Research Articles

Behenic acid protects the testosterone cycle and prevents the sperm apoptosis and protein loss in phthalate exposure by inhibiting oxidative stress and stimulating ATPase activity

BackgroundPlastic products use phthalate to enhance their flexibility, transparency, and stability, while behenic acid is a carboxylic acid with antioxidant activity. ObjectivesThis study evaluates whether behenic acid can protect the testosterone cycle and prevent the sperm apoptosis and protein loss in phthalate-treated male rats. MethodsThere were 36 male albino rats in all, divided into six equal sets of six rats each: control, behenic acid (13g/kg), behenic acid (26g/kg), diethyl phthalate (10mg/kg), behenic acid (13g/kg) + diethyl phthalate (10mg/kg), and behenic acid (26g/kg) + diethyl phthalate (10mg/kg)-treated groups. Measurements were made of serum male hormones, sex hormone-binding globulin, sodium/potassium ATPase, superoxide dismutase, glutathione, glucose-6-phosphate dehydrogenase, 3β-hydroxysteroid dehydrogenase, protein, and cholesterol in the testis, as well as malondialdehyde in the sperm, testis, and hypothalamus. Sperm monoclonal proliferating antibody Ki-67, sperm counts, motility, and abnormalities were measured. ResultsOral administration of diethyl phthalate increased malondialdehyde, serum follicle stimulating hormone, sex hormone binding globulin, luteinizing hormone, glucose-6-phosphate dehydrogenase, 3β-hydroxysteroid dehydrogenase, cholesterol, total protein, sperm abnormality, and the percentage of spermatogonia, first spermatocyte, second spermatocyte, and spermatid in the testis. Superoxide dismutase, glutathione, serum testosterone and dehydroepiandrosterone sulphate, sperm count and motility, and sodium/potassium-ATPase activity were all reduced. Additionally, all of the previously described parameters reverted to near control values after receiving two doses of behenic acid in phthalate-treated rats; a higher dose of behenic acid had a more effective effect than a lower dose. Conclusionbehenic acid can protect the testosterone cycle and prevent the sperm apoptosis and protein loss in phthalate-treated male rats by inhibiting oxidative stress and stimulating ATPase activity.

CD34 and Ki-67 Immunoexpression in Periapical Granulomas: Implications for Angiogenesis and Cellular Proliferation.

The main mechanism of the formation of granulation tissue is the progression of an infection from the tooth to the periapical bone. At this level, the immune system tries to localize and annihilate the microorganism's injury. Ki-67 is a protein directly associated with the cell proliferation rate, while CD34 is a biomarker involved in angiogenesis, and studies suggest that they both have a positive correlation with the intensity of the local inflammatory infiltrate. This study will determine the immunoexpression of CD34 and Ki-67 in periapical granulomas and assess their impact on the growth and development of this tissue, as well as consider their roles in the proliferative process and aggressiveness of evolution. In the present study, 35 periapical granulomas obtained after a tooth extraction were included. The specimens were analyzed via histopathology and immunohistochemistry. A positive reaction for the Ki-67 antibody was observed in 32 (86.5%) of the 35 periapical granuloma cases included in our study. We identified the overexpression of Ki-67 and CD34 and further calculated the Ki-67 index to evaluate and correlate the proliferation potential and angiogenesis with regard to the presence of an inflammatory infiltrate. These findings suggest that the persistence of an inflammatory environment directly influences Ki-67 and CD34 expression, sustaining the proliferative capacity of cells and abnormal angiogenesis. This study is the first to evaluate the presence of the CD34+ and Ki-67+ proliferating vessels in periapical granulomas.

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

Remineralizing capacity of zinc oxide eugenol sealer following the addition of nanohydroxyapatite-tyrosine amino acid: An in vivo animal study

ObjectiveAlthough several sealers have been developed, the zinc oxide eugenol (ZOE) sealer is still used in many private dental clinics. This study aimed to compare the biocompatibility and remineralizing capacity οf ZOE sealer with the addition of nanohydroxyapatite-tyrosine amino acid. MethodsThis study was conducted on Twenty rabbits. The rabbits were divided into four groups based on the test observation period (3, 7, 21, and 28 days) following surgical implantation. General anesthesia was administered to each rabbit, and a subcutaneous incision of approximately 1 ± 0.5 cm was made along the symphyseal area of the mandible of each rabbit. Four bone cavities were generated in the interdental space of the lower jaw between the central and molar teeth of each rabbit, with one longitudinal subcutaneous incision. The ZOE sealers were mixed according to the manufacturer's guidelines and directly inserted within the cavities generated at the end of each test period. The animals were sacrificed, and bone biopsy was carried out at the site of testing. The biopsy samples were obtained and subjected to histological analysis using a low-power light microscope (Olympus C ⅹ 21, Japan) and immunohistochemistry using Ki67 antibody. ResultsThe collected information was examined by parametric statistical tests using SPSS software version ‘‘22.” One-way ANOVA and post-hoc Duncan's tests were used to measure the significance among various groups, with statistical significance set, when “P ≤ 0.01”. ConclusionThe 20% mixed endodontic sealer displayed excellent outcomes compared to other experimental groups, as identified by higher new bone formation at every evaluation period.

Features of beta cell differentiation during the development of type 2 diabetes mellitus

Type 2 diabetes mellitus is characterized by a mild inflammatory reaction in the pancreas, which affects the structure and function of the pancreatic islets: the number of β-cells decreases and the number of α-cells increases. The work examined the features of β-cell differentiation in the development of experimental type 2 diabetes mellitus and while reducing the inflammatory process. Biochemical, histological methods, enzyme-linked immunosorbent assay, immunohistochemical methods were used using primary antibodies to insulin, glucagon, proliferation marker Ki-67 and secondary antibodies labeled with fluorescent dyes. Streptozotocin and nicotinamide were used to model type 2 diabetes mellitus, and the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione was used to reduce the inflammatory response. Previous studies have shown that it changes the macrophage phenotype from proinflammatory M1 to anti-inflammatory M2. In type 2 diabetes mellitus, against the background of a decrease in the number of macrophages with the CD163 marker and the concentration of the cytokine TGF-β1, which have an anti-inflammatory effect, in the pancreatic islets, a decrease in the number of β-cells and their functional activity was observed, while the content of α-cells synthesizing glucagon increased. After administration of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione, the opposite picture was observed in the pancreatic islets: against the background of an increase in the number of CD163+ macrophages and the content of TGF-β1, the number of β cells increased and the number of α cells decreased-cells. The increase in the number of insulin-synthesizing cells was not accompanied by their mitotic activity. It is likely that a decrease in the number of CD163+ macrophages and the level of the antiinflammatory cytokine TGF-β1 in the islets are factors contributing to changes in the cell microenvironment and, as a consequence, the differentiation of β-cells into α-cells. On the contrary, an increase in the number of CD163+ macrophages and TGF-β1 against the background of administration of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione presumably promotes reverse differentiation of α-cells into β-cells and restoration of insulin synthesis pancreas. Targeted effects on the microenvironment of cells in the pancreatic islet in type 2 diabetes mellitus may be a new approach to treating the disease.

Diagnostic Values of Ki67, Cox2, CD31, E-cadherin, VEGF, MMP2, and MMP9 Protein Expression in Human Melanoma

Background: Cutaneous melanoma, the most severe form of skin cancer, has an unpredictable behavior and a high mortality rate. Recent research has identified new biomarkers and their associations with certain histopathological features, offering essential insights into diagnosis, prognosis, and therapeutic strategies. Material and method: In this meticulously designed and executed study, we analyzed 85 formalin-fixed, paraffin-embedded (FFPE) tissue samples using the gold standard technique of immunohistochemistry (IHC) to characterize the protein expression profiles. We used a comprehensive panel of Ki67, Cox2, CD31, VEGF, MMP2, MMP9, and E-cadherin antibodies. Clinical stages were meticulously determined according to TNM classification, the thickness of the Breslow depth, and Clark levels. We employed robust statistical methods such as one-way ANOVA and curve estimation regression to analyze the data. The STRING database, a trusted resource, was used to identify protein-protein interactions and related pathways. Results: The results of our study unveiled novel pairwise positive correlations between CD31 and Ki67 (r = 0.72), and between VEGF and Ki67 protein expression (r = 0.63). We also found a significant inverse relationship between the expression of E-cadherin and some biomarkers, such as Ki67, CD31, and VEGF (p < 0.001 for all). Additionally, our findings revealed a strong positive association between the extent of tumor invasion and the expression levels of Ki67, CD31, and VEGF. The overexpression of these proteins was significantly correlated with advanced clinical stages (p < 0.001 for all). Conclusion: Our study suggests that the biomarkers we examined could be reliable potential diagnostic and prognostic biomarkers for cutaneous melanoma. These findings have the potential to significantly impact the diagnosis, prognosis, and treatment of this deadly disease, offering new avenues for improved patient care.

Sebaceous gland epithelioma with potential malignancy in a dog

Sebaceous gland tumors consist of neoplastic proliferations of sebaceous gland cells located around hair follicles in the dermis. These tumors are subclassified as sebaceous epithelioma, sebaceous adenoma, sebaceous adenocarcinoma and nodular hyperplasia. In this case report, a nodular growth in the sacral region of an eight-year-old male Belgian Malinois dog was presented. Macroscopically, the nodular mass had a slightly soft consistency, grayish-white, and dark red-black appearance. The mass was measured in the dimension of 2x1x1 cm. Histopathologically, the tumor was observed to have a multilobular structure shaped by neoplastic cell islands. The tumor consisted mainly of eosinophilic cells with small cytoplasm resembling epithelial basaloid cells and to a lesser extent differentiated sebocytes. The parenchyma of the tumor consisted of irregular islets containing a small number of mature sebocytes. Their nuclei are oval with one to three small nucleoli. Immunohistochemically, tumor cells for Ki67 antibody showed strongly positive immunoreactivity. Based on the histopathological and immunohistochemical features, the tumor was diagnosed as sebaceous epithelioma having, a potential malignancy. Since no case of sebaceous epithelioma with malignant potential has been reported in our country, we aimed to present the case with histopathological and immunohistochemical features.

Морфофункциональное состояние органов иммунной системы у поросят-гипотрофиков в ранний неонатальный период

Abstract. The purpose of the study was to study the state of the immune system organs in hypotrophic piglets in the early neonatal period in an industrial pig breeding complex. Methods. The experiment was conducted in 2023 in a large industrial pig farm in the Voronezh region on piglets of the wounded neonatal period received from sows 3-4 farrowing. The sows were kept at optimal microclimate parameters, taking into account their physiological state, and fed with SK-1 nutritionally balanced feed. At the initial stage of the experiment, the piglets obtained during farrowing underwent clinical examination and weighing. Animals under 800 g are counted as hypotrophic piglets (n = 30), animals over 800 g are normotrophic, respectively (n =30). After the groups were formed, animals were forced to be slaughtered before taking colostrum (n =5) and biological material was taken from each group (thymus, lymph nodes (inguinal), spleen for immunohistochemical studies. Results. The level of mitotic activity in the thymus of normotrophic piglets was 9.4 % higher (p < 0.05) than in piglets with body weight deficiency. In the spleen, the level of mitotic activity had no significant differences and was approximately the same in all animals participating in the experiment. In lymph nodes, the mitotic activity of cells in normotrophics was 12.7 % higher (p < 0.05). A study of positively expressed CD-3 cells in the spleen revealed significant differences, so, in normotrophic piglets, the number of cells positively stained with this marker was significantly higher than in piglets with a body weight deficiency by 10.2 % (p < 0.05). The number of CD-3 cells in the thymus differed by 5.6 %, and in the lymph nodes by 2.4 % between the groups, but there were no significant differences. The cellular expression of “immature” forms of B lymphocytes (PAX-5) in the lymph nodes of normotrophics was significantly higher by 12.9 % (p < 0.05) compared with hypotrophic piglets. The scientific novelty lies in the fact that for the first time a comprehensive immunohistochemical study of the organs of the immune system of piglets in the early neonatal period was carried out using monoclanal antibodies CD-3, Ki-67 and PAX-5. As a result of the experiment, it was revealed that piglets with body weight deficiency have a “depression” of the immune system, which manifests itself in hypoplasia of T-lymphocytes in the spleen, “maturing” B-lymphocytes in the lymph nodes and low mitotic potential in the thymus and lymph nodes.

Mechanism of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata herbal pair in promoting hepatocellular regeneration in chronic acute liver failure based on HGF/PI3K/Akt signaling axis

Based on the hepatocyte growth factor(HGF)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling axis, this study investigated the therapeutic effect of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata(PRR-ALRP) her-bal pair on acute-on-chronic liver failure(ACLF) rats and its impact on hepatocellular regeneration. The rat model of ACLF was constructed by subcutaneous and tail vein injection of bovine serum albumin combined with intraperitoneal injection of lipopolysaccharides(LPS)+D-galactosamine(D-GalN). The rats were divided into normal control(NC) group, model(vehicle) group, PRR-ALRP(5.85 g·kg~(-1)) group, and hepatocyte growth factor granules(HGFG, 4.05 g·kg~(-1)) group. Hematoxylin-eosin(HE) staining was used to observe pathological changes in rat liver tissues. Serum alanine aminotransferase(ALT), aspartate transaminase(AST), and total bilirubin(TBIL) were detected using an automatic biochemical analyzer. The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) inflammatory factors were detected by ELISA. Immunofluorescence staining was used to detect the positive expression of proliferating cell nuclear antigen(PCNA), antigen identified by monoclonal antibody(Ki67), and cell cycle protein B1(CyclinB1). Real-time fluorescence-based quantitative polymerase chain reaction(RT-qPCR) and Western blot were used to detect the mRNA and protein expression levels of HGF, growth factor receptor-bound protein 1(Gab1), PI3K, Akt, phosphorylated PI3K(p-PI3K), and phosphorylated Akt(p-Akt). The results showed that compared with the vehicle group, the PRR-ALRP group had reduced liver tissue pathological scores, improved liver function, and reduced inflammatory response, with enhanced PCNA, Ki67, and CyclinB1 fluorescence expression. Furthermore, compared with the model group, the PRR-ALRP group showed upregulated expression of HGF and Gab1 proteins, as well as activation of PI3K and Akt phosphorylation. These findings suggest that PRR-ALRP herbal pair exerts anti-liver failure effects by alleviating hepatocyte inflammatory damage and promoting hepatocellular regeneration, and its specific regulatory mechanism may be related to the activation of the HGF/PI3K/Akt signaling pathway.

Effects of Hermetia illucens larvae meal and astaxanthin on intestinal histology and expression of tight junction proteins in weaned piglets.

The weaning phase in piglets causes significant physiological stress, disrupts intestinal integrity and reduces productivity, necessitating strategies to improve intestinal health and nutrient absorption. While current research highlights the role of diet in mitigating these adverse effects, identifying effective dietary supplements remains a challenge. This study evaluated the effects of Hermetia illucens (HI) larvae meal and astaxanthin (AST) on the intestinal histology of weaned piglets. In a controlled experiment, 48 weaned piglets were divided into six groups and received varying levels of HI larval meal (2.5% and 5%) and AST in their diets. The methodology involved comprehensive histological examinations of the small intestine, assessing absorption area, villi elongation, crypt depth, goblet cells, enterocytes and expression of ileal tight junction (TJ) proteins. The study found that HI larval meal significantly improved nutrient absorption in the jejunum and ileum (p < 0.001), thereby enhancing feed conversion. AST supplementation increased the number of enterocytes (p < 0.001). Both HI larval meal and AST positively affected intestinal morphology and function, increasing muscularis muscle mass and villi elongation (p < 0.001 and p < 0.05, respectively). The 2.5% HI meal improved the villi length to crypt depth ratio and slightly increased the goblet cell count (both p < 0.05). Ki-67 antibody analysis showed increased cell proliferation in the duodenal and jejunal crypts, particularly with the 2.5% HI meal (p < 0.001). Insect meal did not affect TJ protein expression, indicating that it had no effect on intestinal permeability. These findings suggest that HI larval meal and AST can enhance the intestinal wellness and productivity of weaned piglets.

PRAME and Historical Immunohistochemical Antibodies Ki-67, P16, and HMB-45 in Ambiguous Melanocytic Tumors.

Ambiguous melanocytic lesions/tumors (AMLs) can be simply described as melanocytic neoplasms that cannot be differentiated as either a melanoma or a nevus. Preferentially expressed antigen in melanoma (PRAME) is a novel antibody that can help differentiate between nevi and melanomas. However, its usefulness remains controversial in AMLs. The aim of this study was to demonstrate the importance of PRAME and diagnostic auxiliary antibodies (Ki-67, p16, HMB-45) in the diagnosis of melanocytic lesions, especially in AMLs. This study included 52 ambiguous melanocytic lesions, 40 nevi, and 40 melanomas. All immunohistochemical studies were performed automatically using the Universal Alkaline Phosphatase Red Detection Kit. Different analytic approaches were used for each antibody based on the literature. Statistically, the multinomial forward stepwise elimination logistic regression analysis was used to create a statistical model to predict the diagnosis of melanocytic lesions based on clinical, morphological, and immunohistochemical data. PRAME positivity was very strong and diffuse in the melanoma group and statistically significantly higher than that of the AML and nevus groups. There was no statistically significant difference between the nevus and AML groups. The Ki-67 proliferation index and HMB-45 staining pattern provided valuable indications for distinguishing between these 3 groups. The P16 antibody was limited in supporting the differential diagnosis. Our statistical model showed that a high mitosis count, central pagetoid spread, and PRAME positivity increased the probability of melanoma against an AML diagnosis. This study showed the advantages of evaluating the PRAME antibody together with morphological features and other immunohistochemical markers (Ki-67 and HMB-45) in the differential diagnosis of melanocytic lesions.

The effects of enoxaparin treatment in a xenograft mouse model of oral squamous cell carcinoma: A pilot study.

Recent studies suggest that enoxaparin may have therapeutic effects on oral squamous cell carcinoma. We aimed to assess this effect utilizing xenograft mouse model through evaluations of proliferation and angiogenesis markers at the RNA and protein levels. Mice were divided into enoxaparin treatment (n = 4), positive control (n = 4) and negative control (n = 3) groups. Immunohistochemical analyses were performed utilizing Bcl-2, Bax and Ki-67 antibodies. Expression levels of proliferation and apoptosis related genes were calculated utilizing qRT-PCR. Time-dependent proliferation assays were performed in OSC-19 and HEK293 cell-lines. Bax antibody showed positive staining in the cytoplasm and nuclei of tumor cells, while Bcl-2 antibody displayed staining only in the cytoplasm. A proliferation index of 15%-20% was found in all groups with the Ki-67 marker indicating no metastasis. Enoxaparin treatment caused decrease in BCL2, BAX and CCNB1 genes' expressions. Compared to HEK293, proliferation assays demonstrated higher division rates in OSC-19 with a significant decrease in viability after 96 h. Reduced BCL-2 expression indicates a regression of tumor growth, but reduced BAX expression is not correlated with increased apoptosis. Despite the aggressive nature of OSC-19, our results showed a low cell viability with a high division rate when compared with the control HEK293. This paralleled our in vivo findings that showed absence of lymph node metastasis across all mice groups. This discrepancy with the literature suggests that further investigations of the underlying mechanisms and protein-level analyses are needed to draw definitive conclusions about the effect of enoxaparin on OSC-19 behavior.

Tumor-matched and unmatched cancer associated fibroblasts exhibit differential effect on proliferation and FMOD and MMP9 gene expression in head and neck squamous cell carcinoma cells when cocultured in spheroids

BackgroundCancer-associated fibroblasts (CAFs) are the major cellular component of the tumor microenvironment and are known to affect tumor growth and response to various treatments. This study was undertaken to investigate the crosstalk between tumor-matched or unmatched CAFs and head and neck squamous cell carcinoma (HNSCC) cells regarding tumor growth and treatment response.MethodsThree HNSCC cell lines (LK0412, LK0902 and LK0923), were cocultured in 2D or in 3D with their tumor-matched CAFs, site matched CAFs from other tumors or normal oral fibroblasts (NOFs). Cell proliferation was assessed as the amount of Ki67 positive cells/ spheroid area in formalin-fixed- paraffin-embedded 3D spheroids stained with Ki67 antibody. Viability after seven days of cisplatin treatment was measured with CellTiter-Glo 3D Viability Assay. The mRNA expression of CAF-associated markers (ACTA2, COL1A2, FAP, PDGFRα, PDGFRβ, PDPN, POSTN and S100A4) in CAFs before and after coculture with tumor cells as well as mRNA expression of CAF-induced genes (MMP1, MMP9 and FMOD) in tumor cells separated from CAFs after co-culture was measured with RT-qPCR. The expression of selected protein biomarkers was validated with immunohistochemistry based on previous mRNA expression results.ResultsThe proliferation of the LK0412 and LK0902 tumor spheroids varied significantly when cocultured with different CAFs and NOFs as shown by Ki-67 positive cells. RT‒qPCR analysis revealed different molecular profile of the analyzed HNSCC-derived CAFs concerning the expression of CAF-associated markers. The interaction between CAFs and HNSCC cells was more pronounced after coculture with unmatched CAFs as shown by changes in mRNA expression pattern of CAF-specific markers. Additionally, the unmatched CAFs significantly upregulated the mRNA expression of MMP1, MMP9 and FMOD in tumor cells compared to tumor-matched CAFs.ConclusionOur results indicate that tumor-matched CAFs are unique for each tumor and affect the proliferation and the gene/protein expression of tumor cells in a distinct manner. The interaction between tumor unmatched CAFs and HNSCC cells in the tumor spheroids is associated with significant changes in the mRNA expression of CAF-specific markers and significant increases in FMOD and MMP9 in tumor cells compared to when cocultured with tumor-matched CAFs. Taken together, our results show how important the selection of CAFs is to get a reliable in vitro model that mimics the patients’ tumor.

Dendritic cell subpopulations in carcinoma ex-pleomorphic adenoma: A multicenter study.

Carcinoma ex-pleomorphic adenoma (CEXPA) represents a malignant transformation from a recurrent or primary pleomorphic adenoma (PA), and the immune response may be essential in this process. Therefore, in this study, we aimed to identify and quantify subpopulations of dendritic cells (DCs) in CEXPA, residual PA in CEXPA (rPA), and PA. A multicenter study was performed collecting salivary gland tumor (SGT) samples from three Oral and Maxillofacial Pathology Centers. A tissue microarray containing 41 samples of CEXPA and 22 samples of PA was included in this study and submitted to immunohistochemical reactions against CD1a, CD83, CD207, and Ki67 antibodies. Both PA and rPA showed a higher quantification of CD207+ and CD83+ cells when compared to CEXPA (p < 0.001 and p < 0.01, respectively). There was also a difference when comparing the cell proliferation index between PA/rPA and CEXPA using the Ki-67 marker (p = 0.043). However, there was no difference in the DC population regarding clinical parameters such as sex, anatomical location, size, and metastases (p > 0.06). Immunohistochemical profile of DC subpopulations and cell proliferation biomarkers in SGTs can contribute as an important tool in the differentiation of benign and malignant tumors or detection of initial areas with malignant transformation.

Histopathologic Changes after Yttrium-90 Radioembolization of Colorectal Liver Metastases: A Pilot Feasibility Study

PurposeTo evaluate the histopathologic changes and potential correlations of tumor absorbed dose (TAD) after yttrium-90 transarterial radioembolization (TARE) for colorectal liver metastases (CLMs). Materials and MethodsThis prospective pilot study assessed 12 patients with 13 CLMs through positron emission tomography (PET)/computed tomography (CT)–guided biopsies before, immediately after TARE (T0), and 3 weeks after TARE (T3). Subsequent sampling from the same location was enabled by fiducial placement. Biopsy samples were evaluated with hematoxylin and eosin, TUNEL, Ki67, OxPhos, caspase-3 (CC3), and pH2AX antibodies. Proliferation changes (Ki67) and double-strand DNA breaks (DSBs) were evaluated quantitatively. TAD was calculated on post-TARE PET/CT scan of the biopsy needle location at T0 and T3. ResultsMedian TAD at 3 weeks after TARE was 162 Gy (interquartile range (IQR), 92–211 Gy). DSBs decreased significantly from T0 (median, 77%; IQR, 75%–100%) to T3 (median, 14%; IQR, 0%–54%; P = .028). A decrease in Ki67 was also documented (median, 73%; IQR, 70%–80% at T0 vs median, 41%; IQR, 0%–66% at T3; P = .046). There was a strong positive correlation between TAD and DSBs at T0 (r[9] = 0.68) and a strong negative correlation at T3 (r[10] = −0.855; P = .042 and P = .002, respectively). There was a strong negative correlation between TAD and Ki67 at both T0 (r[9] = −0.733; P = .025) and T3 (r[10] = −0.681; P = .030). Tumors that exhibited caspase-3 activation (8/13, 62%) at either T0 or T3 time point were more likely to develop progression (7/8 [88%] vs 1/5 [20%]; P = .015). ConclusionsPost-TARE biopsy can be used to assess TAD and histopathologic changes. Significant decreases in DSBs and proliferation index were noted after TARE. Post-TARE CC3 activation deserves further exploration.

Hepatoprotective perspective of newly synthesized 3-(3,5-bis (tri fluoro methyl) phenyl)- 5-methyl-1-((1-methyl-1h-pyrrol-2-yl) methyl)-2-thioxoimidazolidin-4-one against diethyl nitrosamine induced liver injury in rats with molecular docking investigatio

The hepatoprotective effect of synthesized 3-(3,5-bis (trifluoromethyl) phenyl)-5-methyl-1-((1-methyl-1H-pyrrol-2-yl) methyl)-2-thioxoimidazolidin-4-one (3FL-M) was evaluated against diethyl nitrosamine-induced liver injury (DEN). Wistar rats were divided into 3 groups as: placebo (received 10% tween 80%), hepatotoxic control (injected with 200 mg/kg of DEN) and treatment (injected 200 mg/kg of DEN and received 50 mg/kg oral feeding of the synthesized 3FL-M). Half the number of the rats were sacrificed on 2nd week of the experiment, whereas the other half were sacrificed after 6 weeks. Blood was collected to run liver biochemical analysis, and to evaluate pro-inflammatory cytokines tumor necrosis factor-alpha TNF-α and interleukine6 IL-6. Liver sections were used to detect nuclear protein ki-67 and hepatocyte specific antibody HSA. 3FL-M was subjected to molecular docking calculations based on binding affinities towards TNF-α and IL-6. DEN-treated rats showed elevation in the liver serum enzymes as well as pro-inflammatory cytokines with clear destruction of the hepatic architecture, in contrast 3FL-M treated rats showed normalized liver enzymes and cytokines levels with resolution of the hepatocytes. Molecular modelling revealed that 3FL-M exhibited the significant affinities toward the binding pocket of the TNF-α and IL-6, however, further studies is recommended for developing it as a chemotherapeutic drug-like molecule.&#x0D; KEY WORDS: DEN, Hepatoprotective, TNF-α, IL-6, Molecular docking&#x0D; Bull. Chem. Soc. Ethiop. 2024, 38(3), 751-763. &#x0D; DOI: https://dx.doi.org/10.4314/bcse.v38i3.16

Histopathologic and Immunohistochemical Analysis of Neurofibromas in a North-Western Nigerian Tertiary Hospital: A Ten-Year Retrospective Study.

Neurofibromas are the most common benign nerve sheath tumours occurring as solitary sporadic tumours or multiple Syndromic tumours associated with neurofibromatosis type 1(NF1). In Nigeria and West Africa, there is a paucity of literature and studies on neurofibromas. This study aims to analyse the histopathologic and immunohistochemical patterns of neurofibromas and determine the frequency, demographic and anatomic distributions. The study was a hospital-based retrospective study, and the study population constituted all surgical specimens submitted for histological examination to the Department of Pathology between 1st January 2010 to 31st December 2019 reported as neurofibroma. Records were retrieved from the archives and subjected to histopathologic and immunohistochemical analysis following standard protocols. Collated data was analysed, slides were reviewed, and results were presented in frequency distribution tables and statistical charts. A total of 125 cases were seen constituting 8.3% of all soft tissue tumours seen. Neurofibromas were more prevalent in females with a male-to-female ratio of 1:1.15. The age ranged between 2-70 years with a mean age of 25.38 years and the highest frequency of occurrence was in the second decade of life. The most frequent anatomic site of occurrence was the head and neck region. Most of the tumours 103 (82.4%) were sporadic while 22(17.6%) were Syndromic and associated with NF1. A malignant transformation of a pre-existing neurofibroma in an NF1 patient was seen. The most common histologic variant seen was the conventional variant. Ninety percent of these tumours showed SOX10 immunopositivity, 91% showed S100 immunopositivity and 95% showed CD34 immunopositivity. Calretinin expression was low showing 16%. No hot spots labeling index seen with Ki67 antibody. Neurofibromas are more common in females in our environment and the most frequent anatomic site of involvement is the head and neck region.

Does YAP influence cell proliferation and apoptosis in benign epithelial odontogenic lesions?

To analyze the immunohistochemical expression of YAP and its correlation with markers involved in cell proliferation and apoptosis in benign epithelial odontogenic lesions. The sample consisted of 95 cases of odontogenic lesions (25 dentigerous cysts, 30 non-syndromic odontogenic keratocysts, 30 conventional ameloblastomas, and 10 unicystic ameloblastomas) and 10 dental follicles used as normal odontogenic tissue. The histological sections were submitted to immunohistochemistry with YAP, cyclin D1, Ki-67, and Bcl-2 antibodies. Immunoexpression was analyzed qualitatively and quantitatively using an adapted method. The collected data were analyzed descriptively and statistically (p ≤ 0.05). The highest YAP expression was observed in odontogenic keratocysts, followed by unicystic ameloblastomas and conventional ameloblastomas, which exhibited moderate immunoreactivity predominantly in peripheral cells. Furthermore, significant differences in YAP immunoexpression were observed between the groups analyzed, with significant positive correlations between YAP and cyclin D1 in dentigerous cysts and unicystic ameloblastomas and between YAP and Ki-67 in unicystic ameloblastomas (p < 0.05). However, there were no statistically significant correlations between YAP and Bcl-2 immunoexpression in the groups studied. YAP may influence epithelial cell proliferation in odontogenic cysts and tumors, suggesting its possible participation in the progression of the odontogenic lesions studied.

Correlation between PD-L1 and Ki-67 Expression at various T-stage Clear Cell Renal Cell Carcinomas

Renal cell carcinoma (RCC) is a malignant neoplasm originating from renal epithelium, with the clear cell renal cell carcinoma (ccRCC)being the most common type (80%) and the most common cause of death among other types of kidney cancer. Pathological stage is an important parameter that affects ccRCC survival, followed by nuclear grade. Pathological staging of RCC according to the AJCC (American Joint Committee on Cancer) TNM system 8th edition is based on local extension of the main tumor (T), involvement of lymph node (N), and metastasis (M). Ki-67 is a marker of proliferation used to assess tumor grade. High Ki-67 correlates with poor prognosis, advanced clinical and pathological features, thus Ki-67 can be used as a biomarker in the management of RCC.Ki-67 is routinely used to see the proliferation index in various cases of malignancy, but not in kidney malignancy. Programmed death ligand 1 (PD-L1) acts as a negative regulator of T cell-mediated anti-tumor immune responses. PD-L1 is expressed on T cells, B cells, macrophages, dendritic cells, endothelial cells and in various tumor cells including ccRCC. This study aims to determine the correlation between the expression of PD-L1 and Ki-67 in various T-stage clear cell renal cell carcinomas. Material and Method: This was an observational analytical study with cross-sectional approach toward 52 cases of ccRCC whose diagnosis was made histopathologically at the Anatomical Pathology Installation of Dr. Soetomo General Academic Hospital Surabaya from January 2014 to December 2020. Immunohistochemical stainingwas carried out using Ki-67 and PD-L1 antibodies, followed by an assessment using a scoring system. T-stage data were obtained from the patients’ medical recordswhich were then analyzed statistically with the Spearman test. Result: The study included 52 cases of ccRCC obtained from nephrectomy specimens at RSUD dr. Soetomo between 2014–2020. The age distribution of the subjects was 29–69 years and the mean and median age was 53 years. The ratio of male patients compared to female patients was 2.5:1. The majority was stage T2 (50%). Statistical test results showed no correlation between the expression of PD-L1 and Ki-67 in various T-stage clear cell renal cell carcinomas (p=0.965 and p=0.680). Conclusion: This study showed no correlation between the expression of PD-L1 and Ki-67 in various T-stage clear cell renal cell carcinomas. Nonetheless PD-L1 can be considered as an important biomarker with a poorer prognosis and aggressive clinicopathological findings in patients with RCC.