Preclinical Study in Mouse Thymus and Thymocytes: Effects of Treatment with a Combination of Sodium Dichloroacetate and Sodium Valproate on Infectious Inflammation Pathways.

The research presents data from a preclinical study on the anti-inflammatory effects of a sodium dichloroacetate and sodium valproate combination (DCA-VPA). The 2-week treatment with a DCA 100 mg/kg/day and VPA 150 mg/kg/day combination solution in drinking water's effects on the thymus weight, its cortex/medulla ratio, Hassall's corpuscles (HCs) number in the thymus medulla, and the expression of inflammatory and immune-response-related genes in thymocytes of male Balb/c mice were studied. Two groups of mice aged 6-7 weeks were investigated: a control (n = 12) and a DCA-VPA-treated group (n = 12). The treatment did not affect the body weight gain (p > 0.05), the thymus weight (p > 0.05), the cortical/medulla ratio (p > 0.05), or the number of HCs (p > 0.05). Treatment significantly increased the Slc5a8 gene expression by 2.1-fold (p < 0.05). Gene sequence analysis revealed a significant effect on the expression of inflammation-related genes in thymocytes by significantly altering the expression of several genes related to the cytokine activity pathway, the inflammatory response pathway, and the Il17 signaling pathway in thymocytes. Data suggest that DCA-VPA exerts an anti-inflammatory effect by inhibiting the inflammatory mechanisms in the mouse thymocytes.

Tumors derived from lung cancer cells respond differently to treatment with sodium valproate (a HDAC inhibitor) in a chicken embryo chorioallantoic membrane model.

Lung cancer is the most frequent cause of cancer death. Some human lung malignant tumors have a combined small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) histology, with tumor cell phenotype changing during tumor progression. Valproic acid is used as an anti-seizure medication to treat migraine, and bipolar mood disorders. Recently, its efficacy as an adjuvant therapy was shown in cancer due to its histone deacetylase (HDAC) inhibitory property. HDACs are upregulated in lung tumors, and HDAC inhibitors, including valproic acid, inhibit endothelial cell proliferation in vitro and in vivo and have antiproliferative and antimigratory properties. We tested valproic acid for possible antiangiogenic and antimigratory effects on experimental lung tumors grafted onto the chicken embryo chorioallantoic membrane (CAM). Tumors were formed from two NSCLC cell lines and a single SCLC cell line. To investigate tumor and CAM interactions, in vivo biomicroscopy, visualization of blood vessels with injected fluorescent dextran, histological, immunohistochemical and histomorphometric methods were applied. Our results showed that a sodium valproate (NaVP) treatment-induced a dose-dependent decrease of experimental tumor invasion into the CAM mesenchyme and a reduction in angiogenesis. Both the invasion and the angiogenic response were dependent on the type of cell line used: invasion and angiogenesis of tumors derived from A549 and NCI-H146 cell lines responded to increasing doses of NaVP from 4 to 8 mM, whereas Sk_Lu_1 cells response were antimigratory and antiangiogenic when NaVP was used up to 6 mM. When 8mM NaVP was used, stimulated invasion and angiogenesis in tumors from Sk_Lu_1 cells were observed.

Laryngeal carcinoma experimental model suggests the possibility of tumor seeding to gastrostomy site.

Some studies state that laryngeal squamous cell carcinoma (LSCC) is associated with possible direct tumor cell seeding to percutaneous endoscopic gastrostomy (PEG) site. However, there is a lack of experimental proof that LSCC tumor tissue can adhere and grow in distant sites. Therefore, we aimed to investigate the growth pattern of LSCC implants on chicken embryo chorioallantoic membrane (CAM) and evaluate possible associations between clinical course of the disease and behavior of experimentally implanted LSCC tumors. Our results show that implanted LSCC tissue survives on CAMs in 95% of cases while retaining essential morphologic characteristics and proliferative capacity of the original tumor. We identified the increased CAM vascularization, an infiltrative growth pattern of the implant and formation of distant isolated metastatic nodes on the CAMs. LSCC tumors with worse differentiation degree (G2 or G3) adhered to the experimental CAMs significantly better than G1. These results facilitate the understanding of tumor biology and allow hypothetisezing that dissemination and direct implantation of LSCC cells into the stomal wall during the pull PEG procedure might be possible.

An Experimental Model of Human Recurrent Respiratory Papillomatosis: A Bridge to Clinical Insights.

OBJECTIVES/HYPOTHESIS: To investigate the growth pattern of recurrent respiratory papilloma (RRP) implants on chicken embryo chorioallantoic membranes (CAMs) and to evaluate possible associations between the clinical course of the disease and the behavior of experimentally implanted RRP tumors. STUDY DESIGN: Experimental study. METHODS: Fresh 172 RRP tissue samples from 12 patients were implanted onto chick embryo CAMs. Morphological and morphometric analysis of the experimental CAM and chorionic epithelium was performed. The microvascular network of the CAM with the RRP implant was investigated under the effect of fluoresceinated anionic dextran. The peculiarities of the clinical course of the disease were evaluated. RESULTS: The implanted RRP tissue samples survived on CAMs in 86% of cases, retaining their essential morphologic characteristics and proliferative capacity of the original tumor. Implants induced thickening of both the CAM and the chorionic epithelium, but none of the RRP implants crossed the basement membrane of the hosting CAM. A "crawling film" of acellular material with newly formed papilloma sprouts located on the outer chorionic epithelium of the CAM was detected. Direct association between a recurrence rate of RRP and the number of newly formed papilloma sprouts around the implanted tumor on CAMs was revealed. CONCLUSION: The chicken embryo CAM-based model is appropriate for investigations of RRP and facilitates the understanding of tumor biology and the clinical course of the disease, thus providing the basis for further research and acceleration of the identification and development of new specific therapeutic compounds that limit the spread and recurrence of RRP. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E914-E920, 2021.

Tumor Suppression in Asymptomatic Postmenopausal Endometrial Polyps.

BACKGROUND/AIM: To investigate tumor suppression as an indicator of malignization potential within endometrial polyps in asymptomatic postmenopausal women. MATERIALS AND METHODS: Immunohistochemical studies of the phosphatase and tensin homolog (PTEN) were performed. Cases included 52 benign postmenopausal polyps, 19 endometrioid carcinomas with coexisting benign polyps, and 12 polyps with foci of carcinoma. Controls included 31 atrophic endometria and 32 benign premenopausal polyps. PTEN was scored by quantitative methods according to staining intensity. RESULTS: The mean epithelial and stromal PTEN H-score in postmenopausal benign endometrial polyps (193.8 and 123.2, respectively) was significantly higher than that in the atrophic endometrium (135.5 and 90.2, p=0.008), and premenopausal benign endometrial polyps (100.7 and 198.7, p<0.001). Significant difference between postmenopausal endometrial polyps and endometrial carcinoma was noticed in the epithelial compartment (193.8 vs. 65.7, respectively, p<0.001). CONCLUSION: Asymptomatic benign postmenopausal polyps have a distinctively high tumor suppression compared with endometrial cancer, suggesting low malignization potential.

Proliferation in Postmenopausal Endometrial Polyps-A Potential for Malignant Transformation.

Background and objectives: Endometrial polyps in asymptomatic postmenopausal women are often incidentally found, yet only 1.51% of them are malignant. Their potential for malignant transformation has not been adequately addressed. The aim of this study was to investigate the proliferation within endometrial polyps as one of the indicators of their malignization potential in asymptomatic postmenopausal women. Materials and Methods: Immunohistochemical studies of Ki-67 were performed. Cases included 52 benign postmenopausal polyps, 19 endometrioid carcinoma with coexisting benign polyps, 12 polyps with foci of carcinoma and 4 cases of polyps, which later developed carcinoma. The control group included 31 atrophic endometria and 32 benign premenopausal polyps. Ki-67 was scored in either 10 or 20 "hot spot" fields, as percentage of positively stained cells. Results: The median epithelial Ki-67 score in postmenopausal benign polyps (4.7%) was significantly higher than in atrophic endometria (2.41%, p < 0.0001) and significantly lower than in premenopausal benign polyps (11.4%, p = 0.003) and endometrial cancer (8.3%, p < 0.0001). Where endometrial polyps were found in association with endometrial carcinoma, Ki-67 was significantly higher in cancer (p < 0.0001). No significant difference was found between Ki-67 scores of cancer focus and of the polyps tissue itself, respectively 2.8% and 4.55%, p = 0.37. Ki-67 expression, where polyps were resected and women later developed cancer, was not significantly different (p = 0.199). Conclusion: Polyps from asymptomatic postmenopausal women showed significantly more proliferation in both epithelial and stromal components than inactive atrophic endometria but less than premenopausal benign polyps and/or endometrial cancer. Benign postmenopausal endometrial polyps exhibit low proliferative activity, suggesting low malignant potential and may not require resection in asymptomatic women.

Sodium Dichloroacetate Pharmacological Effect as Related to Na-K-2Cl Cotransporter Inhibition in Rats.

The study objective was to investigate a possible sodium dichloroacetate (DCA) pharmacological mechanism causing an increase in diuresis in rats. The aim was to define characteristics of 24-hour urinary Na+, K+, Cl-, Ca2+, and Mg2+ excretion in Wistar male rats and to evaluate effect of a single-dose DCA and repeated DCA dosage on diuresis. Six control and 6 DCA-treated male rats aged 5 to weeks after a single DCA dose and repeated dosage were tested. The single DCA dose treatment caused a significantly higher 24-hour diuresis when compared to control (P < .05), and it was related to increased Cl-, Na+, and K+ urine excretion and a significant increase in Ca2+ and Mg2+ excretion (P < .05); after the repeated 4-week DCA dosage, the diuresis was not increased, but the excretion of the Na+, Cl-, Ca2+, and Mg2+ ions was significantly higher. Kidney immunohistochemistry has revealed that DCA continuous treatment results in an increase in the size of Henle loop thick ascending limb epithelial cells (P < .001). The study results show a significantly reduced RNA expression of Na-K-2Cl co-transporter (NKCC1) in thymus of 4-week DCA-treated rats (P < .03). The study data have indicated a possible mechanism of such pharmacological effect to be NKCC inhibition.

Model of human recurrent respiratory papilloma on chicken embryo chorioallantoic membrane for tumor angiogenesis research.

We aimed to develop a chick embryo chorioallantoic membrane (CAM) model of recurrent respiratory papilloma (RPP) and to evaluate its morphological and morphometric characteristics, together with angiogenic features. Fresh RRP tissue samples obtained from 13 patients were implanted in 174 chick embryo CAMs. Morphological, morphometric, and angiogenic changes in the CAM and chorionic epithelium were evaluated up until 7 days after the implantation. Immunohistochemical analysis (34ßE12, Ki-67, MMP-9, PCNA, and Sambucus nigra staining) was performed to detect cytokeratins and endothelial cells and to evaluate proliferative capacity of the RRP before and after implantation on the CAM. The implanted RRP tissue samples survived on CAM in 73% of cases while retaining their essential morphologic characteristics and proliferative capacity of the original tumor. Implants induced thickening of both the CAM (241-560%, p=0.001) and the chorionic epithelium (107-151%, p=0.001), while the number of blood vessels (37-85%, p=0.001) in the CAM increased. The results of the present study confirmed that chick embryo CAM is a relevant host for serving as a medium for RRP fresh tissue implantation. The CAM assay demonstrated the specific RRP tumor growth pattern after implantation and provided the first morphological and morphometric characterization of the RRP CAM model that opens new horizons in studying this disease.

Effect of Laryngeal Squamous Cell Carcinoma Tissue Implantation on the Chick Embryo Chorioallantoic Membrane: Morphometric Measurements and Vascularity.

BACKGROUND: The aim of this study was to develop chick embryo chorioallantoic membrane (CAM) model of laryngeal squamous cell carcinoma (LSCC) and to evaluate the morphological and morphometric characteristics and angiogenic features of it. METHODS: Fresh LSCC tissue samples obtained from 6 patients were implanted onto 15 chick embryo CAMs. Morphological, morphometric, and angiogenic changes in the CAM and chorionic epithelium were evaluated up to 4 days after the tumor implantation. Immunohistochemical analysis (34ßE12, CD31, and Ki67 staining) was performed to detect cytokeratins and tumor endothelial cells and to evaluate the proliferative capacity of the tumor before and after implantation on the CAM. RESULTS: The implanted LSCC tissue samples survived on the CAM in all the experiments and retained the essential morphologic characteristics and proliferative capacity of the original tumor. Implants induced thickening of both the CAM (103-417%, p = 0.0001) and the chorionic epithelium (70-140%, p = 0.0001) and increase in number of blood vessels (75-148%, p = 0.0001) in the CAM. CONCLUSIONS: This study clarifies that chick embryo CAM is a relevant assay for implanting LSCC tissue and provides the first morphological and morphometric characterization of the LSCC CAM model that opens new perspectives to study this disease.

Cardiomyocyte remodeling in ischemic heart disease.

OBJECTIVE: The aim of the study was to detect changes in left ventricular cardiomyocyte size and shape in response to chronic ischemia and loss of cardiac tissue (myocardial infarction) during the course of ischemic heart disease (IHD). MATERIAL AND METHODS: Left ventricular cardiomyocyte dimensions (diameter and length) were estimated histomorphometrically, and their cross-sectional area and volume were assessed in 85 males who died suddenly out of hospital (within 6 hours of the onset of the terminal event) due to the acute first (preinfarction IHD group, n=53, aged 48.6+/-2.9 years) or repeated (postinfarction IHD group, n=32, aged 51.7+/-2.9 years) IHD attack, and had no other causes for the increased heart load. Twenty-nine males of similar age (mean age, 46.0+/-3.1 years) who succumbed to external causes served as controls. RESULTS: We have found cardiomyocyte hypertrophy in the preinfarction IHD group already. The cardiomyocyte volume was increased by 32.0% in comparison with the same index in the control group, and cross-sectional area and length--by 17.2 and 12.5%, respectively. In postinfarction IHD group, all studied cardiomyocyte parameters did not differ significantly from the analogous indices in the preinfarction IHD group (P>0.05). Cardiomyocyte hypertrophy was related to the increase in left ventricular cardiomyocyte parameters. CONCLUSIONS: Left ventricular cardiomyocyte hypertrophy occurs before the first myocardial infarction. In postinfarction myocardium, cardiomyocyte dimensions do not differ significantly at least prior to the appearance of congestive heart failure syndrome.

[Myocardial pathology in sudden ischemic death: peculiarities of morphologic diagnosis]. / Nuo isemines sirdies ligos staiga mirusiuju miokardo patologija: morfologines diagnostikos ypatybes.

OBJECTIVE: The aim of the investigation was to determine morphological criteria of acute myocardial injures in sudden ischemic death. MATERIAL AND METHODS: Morphological investigation of the whole myocardium and coronary arteries in 170 victims of sudden out-of-hospital death due to ischemic heart disease was performed in the framework of the international WHO program "Myocardial Infarction Register in Population" and joint (at that time) USSR-USA project on sudden death. RESULTS: It was established, that out-of-hospital sudden death due to ischemic heart disease in all cases is related to irreversible myocardial injury: 92.9% - to acute myocardial infarction, and 7.1% - to micronecrosis. The following phases of morphological development of infarction were determined: early myocardial infarction--in 48.8, definite--in 21.8, and progressing--in 22.3%. Since the signs of early infarction were also found in 34 cases of progressing myocardial infarction, it was reasonable to suppose that in 117 (71.1%) patients the occurrence of sudden out-of-hospital death due to ischemic heart disease was somehow connected with the very early and early phases of acute myocardial infarction. CONCLUSIONS: Accurate identification of early myocardial infarction is available only by microscopic investigation of histotopograms of the whole myocardium considering the complex of signs of cardiomyocyte alteration as well as early inflammatory reaction. Acute pathology (erosion or rupture of atherosclerotic plaque and thrombosis) of "culprit" coronary artery indirectly indicates regional myocardial irreversible injury.