Assessment of Computational Histopathology in Thoracic Aortic Aneurysm

Background and Hypothesis: Thoracic aortic aneurysm (TAA) histopathology includes elastic fiber (EF) abnormalities, mucoid extracellular matrix (MECM) accumulation, and smooth muscle derangement in the aortic medial layer. While semi-quantitative grading of these characteristics is a standard practice, computational characterization of medial layer components may facilitate novel quantitative analyses at higher throughput. We hypothesized that computational results would correlate with results of semi-quantitative grading of aortic histopathology. Experimental Design: Formalin-fixed, paraffin-embedded human aortic tissue sections were stained with Movat’s pentachrome to characterize aortic microstructure. Sections were also immunostained for nitrotyrosine residues to assess oxidative stress. Samples were initially graded semi-quantitatively by two independent blinded readers. Next, computational histopathology software was used a) to quantify the proportions of EF, MECM, and cellular area in the medial layer of pentachrome-stained sections and b) to quantify the distribution and intensity of positive nitrotyrosine staining in immunostained sections. Association between semi-quantitative grading and computed values was tested with ANOVA. Results: The cohort included 74 participants who underwent prophylactic aortic replacement for TAA and 23 healthy controls. The mean age was 54±17 years. On average, EFs accounted for 49% (range 6-90%) of medial tissue area, whereas MECM accounted for 25% (1-73%). The overall semi-quantitative grade of medial degeneration severity was associated with decrease in EF fraction (p=0.02). The grade of EF thinning also strongly correlated with decrease in EF fraction (p=1x10-6). Meanwhile, grade for accumulation of MECM was associated with increase in MECM (p=0.004). Increased semi-quantitative grading for nitrotyrosine levels was associated with increased nuclear signal optical density (p=9x10-10) and greater percentage of cells labeled as strongly positive (p=8x10-10). Conclusion and Potential Impact: We observed significant correlations between computed quantitative values and semi-quantitative grading. This suggests that computational histopathology is a valid method for investigation of human TAA tissues.

DESIGNER DNA HYDROGELS TO STIMULATE 3D CELL INVASION BY ENHANCED RECEPTOR EXPRESSION AND MEMBRANE ENDOCYTOSIS

DNA has emerged as one of the smartest biopolymers to bridge the gap between chemical science and biology to design scaffolds like hydrogels by physical entanglement or chemical bonding with remarkable properties. We present here a completely new application of DNA based hydrogels in terms of their capacity to stimulate membrane endocytosis, leading to enhanced cell spreading and invasion for cells in ex-vivo 3D spheroids models. Multiscale simulation studies along with DLS data showed that the hydrogel formation was enhanced at lower temperature and it converts to liquid with increase in temperature. DNA hydrogels induced cell spreading as observed by increase in cellular area by almost two-folds followed by increase in receptor expression, endocytosis and 3D invasion potential of migrating cells. Our first results lay the foundation for upcoming diverse applications of hydrogels to probe and program various cellular and physiological processes that can have lasting applications in stem cells programming and regenerative therapeutics.

Increased Caspase-3 Immunoexpression and Morphology Alterations in Oenocytes and Trophocytes of Apis mellifera Larvae Induced by Toxic Secretion of Epormenis Cestri

Toxic honeydew produced by Flatidae Epormenis cestri in Uruguay has been shown to cause among honeybees (Apis mellifera) colonies a massive larva death called “River disease”, but the intrinsic mechanisms are still unknown. Because fat body cells, oenocytes and trophocytes, are known to regulated larvae metabolism, and to be affected by xenobiotics, we tested whether apoptosis of these cells can be an underlying cause of larvae death. Ten colonies were divided into two groups and fed with common honey or toxic honeydew obtained from colonies affected by “River disease”. Five-dayold larvae were collected and processed for histology and immunohistochemistry for caspase-3. The area, diameter, and immunostaining area in oenocytes and trophocytes were measured. The oenocyte and trophocyte cellular area decreased in the treated group (p=0.002; p<0.001 respectively) compared to the control group. The diameter of oenocytes (p=0.0002) and trophocytes (p<0.0001) decreased in the treated group. Caspase-3 was detected in cytoplasm in the control group but in the cytoplasm and nucleus in the treated group. The caspase-3 immunostaining area increased in oenocytes (p<0.002) and trophocytes (p<0.0001) of the treated group. The ingestion of toxic honeydew altered the morphology, localization and immunoexpression of caspase-3 in fat body cells, which suggests that the deregulation of the apoptotic mechanism affected the normal development in A. mellifera larvae.

ATP triggers macropinocytosis that internalizes and is regulated by PANX1

ABSTRACTMacropinocytosis is an endocytic process that allows cells to respond to changes in their environment by internalizing nutrients and cell surface proteins, as well as modulating cell size. Here, we identify that adenosine triphosphate (ATP) triggers macropinocytosis in murine neuroblastoma cells, thereby internalizing the ATP release channel pannexin 1 (PANX1) while concurrently increasing cross-sectional cellular area. Amiloride, a potent inhibitor of macropinocytosis-associated GTPases, abolished ATP-induced PANX1 internalization and cell area expansion. Transient expression of the GTP-hydrolysis resistant GTPase ARF6 Q67L led to increased PANX1 internalization and increased cell area equivalent to levels seen with ATP stimulation. Mutation of an extracellular tryptophan (W74) in PANX1 abolished ATP-evoked cell area enlargement suggesting that PANX1 regulates this form of macropinocytosis. This novel role of PANX1 in macropinocytosis could be particularly important for disease states implicating PANX1, such as cancer, where ATP can act as a purinergic regulator of cell growth/metastasis and as a supplementary energy source following internalization.

Differential diagnosis of chiasmal-cellular cyst

The clinical symptoms of chiasmal-cellular formations are similar, which significantly complicates its differential diagnostics. The differential diagnostics of chiasmal-cellular cysts, which include colloid cysts, arachnoid cysts, Rathke’s pouch cysts, epidermoid and dermoid cysts, is especially difficult. Nevertheless, an accurate preoperative differential diagnostics of chiasmal-cellular cysts is an important stage of preparation for surgical treatment, which allows determining the surgical tactics in advance, because each group of chiasmal-cellular cysts has its own features of surgical treatment, which significantly reduce the number of complications and minimize the number of recurrences. This study intended to improve the efficiency of diagnostics of the chiasmal-cellular cysts by determining the criteria for its differential diagnostics. 94 patients with chiasmal-cellular cysts and pituitary adenomas were examined and treated in the period of 2009 and 2018 for this purpose. As the most frequent pathology of the chiasmal-cellular area, pituitary adenomas were selected as a comparison group due to the fact that it is often necessary to differentiate chiasmal-cellular cysts with this pathology. Patients were divided into 5 groups according to the nosology of the disease. Clinical picture, laboratory analysis and MRI data were studied in each group. Statistical analysis and comparison of the data obtained among all groups were performed, and it allowed to determine the distinctive diagnostic features incidental to each group. It is possible to make an accurate preoperative diagnosis based on the specific features of differential diagnostics.

Interection effects betwen C2C12 and Walker 256 cells

Cancer is the second cause of death in the world and can affect the host metabolism, increasing the muscle spoliation and decreasing the adipocyte tissue, inducing the cachectic state in these patients. This work aimed to analyse how the factors produced by tumour cells could affect the myotube C2C12 growth or activities when these cells were treated with culture medium from tumour cells; with this procedure, we could mimic the same situation as in cancer patient. The results showed that there is a correlation between the cellular area variation and the time of the treatments, and the W group led to a decreased cell confluence layer, suggesting that the tumour cells medium likely contained some factor that affected the C2C12 growth.

A cytomorphometric analysis of the oral mucosa in patients with type 2 diabetes mellitus

Background: Although many of the pathological conditions of oral mucosa are clinically distinguishable, most lesions require a definitive diagnosis. This article tried the use of exfoliative cytology as an alternative tool in the screening, diagnosis and follow-up of diabetes mellitus.Materials and Methods: After rinsing the mouth with normal saline, slides were prepared from buccal mucosa and dorsum of tongue and fixed in 95% ethyl alcohol. The slides were stained with Papanicolaou stain and Acridine orange. Fifty clearly defined cells in each slide were visualized under light microscope for cytomorphometric analysis of cells using Image J software and under fluorescence microscope for assessment of nuclear alterations like micronuclei, nuclear budding, binucleation, multinucleation and karyorrhexis.Results: Statistically significant increase in Nuclear area BM (p = 0.000057), Nuclear Area Tongue (p= 0.0000113), total Nuclear Area (p= 000079), Cellular Area BM (p= 0.0475), Cellular Area Tongue (p= 0.0105), Total Cellular Area (p= 0.00496), Cytoplasmic Area Tongue (p= 0.00358), Total Cytoplasmic Area (p= 0.00268) were obtained from epithelial cells in the diabetic group when compared with the control group. Also the epithelial cells from the diabetic group showed features such as nuclear budding, micronuclei, binucleation, karyorrhexis and perinuclear halo. Conclusion: The objective demonstration of cytomorphometric and nuclear alterations by the oral exfoliated cells indicate the presence of cytological changes in the oral mucosa of diabetic patients despite the apparently normal clinical appearance. Hence, cytomorphometric analysis would aid the health professional as an additional non-invasive tool for the screening and monitoring of Diabetes Mellitus.

Cytomorphometry of serosal effusion in dogs

Cytomorphometry made on cytological slides is the quantitative method of precise analysis of cellular structures, including both cytoplasm and nucleus. The aim of this study was to describe cytomorphometric parameters of mesothelial cells in the course of benign reactive and malignant proliferation and to compare them to carcinomas and adenocarcinomas located within serosal cavities in dogs. The second aim was to evaluate applicability of cytomorphometry to diagnostics of diseases causing accumulation of effusion in serosal cavities. Cytological samples of normal and non-malignant mesothelium, mesothelioma and various carcinomas were collected from dogs. Cytomorphometry was made on the smears stained with Giemsa solution. Mean nuclear and cellular perimeter, mean nuclear and cellular area, mean nuclear and cellular diameter, and mean nuclear and cellular roundness were determined. Moreover, nuclear to cytoplasmic ratio (N/C) was calculated. The data revealed statistically significant differences for all parameters, excluding mean nuclear perimeter, between compared groups. Normal mesothelium cells and their nuclei were significantly smaller and more elongated than cells and nuclei of both benign reactive and malignant neoplastic mesothelium. Only a few differences were observed between benign reactive mesothelium cells and mesothelioma cells – mean nuclear area and mean nuclear diameter of benign reactive mesothelium cells were significantly larger and N/C ratio was higher in comparison to mesothelioma cells. Even though some significant differences were observed, considerable overlap of these cytomorphometric parameters in animals with different diseases limited practical role of these observations. Cytomorphometric analysis of cellular samples collected from dogs with proliferative processes affecting serosal cavities can be only an auxiliary method increasing accuracy of preoperative diagnosis.

IL-1β expression in the distal lung epithelium disrupts lung morphogenesis and epithelial cell differentiation in fetal mice

Perinatal inflammation and the inflammatory cytokine IL-1 can modify lung morphogenesis. To examine the effects of antenatal expression of IL-1β in the distal airway epithelium on fetal lung morphogenesis, we studied lung development and surfactant expression in fetal mice expressing human IL-1β under the control of the surfactant protein (SP)-C promoter. IL-1β-expressing pups suffered respiratory failure and died shortly after birth. IL-1β caused fetal lung inflammation and enhanced the expression of keratinocyte-derived chemokine (KC/CXCL1) and monocyte chemoattractant protein 3 (MCP-3/CCL7), the calgranulins S100A8 and S100A9, the acute-phase protein serum amyloid A3, the chitinase-like proteins Ym1 and Ym2, and pendrin. IL-1β decreased the percentage of the total distal lung area made up of air saccules and the number of air saccules in the lungs of fetal mice. IL-1β inhibited the expression of VEGF-A and its receptors VEGFR-1 and VEGFR-2. The percentage of the cellular area of the distal lung made up of capillaries was decreased in IL-1β-expressing fetal mice. IL-1β suppressed the production of SP-B and pro-SP-C and decreased the amount of phosphatidylcholine and the percentage of palmitic acid in the phosphatidylcholine fraction of lung phospholipids, indicating that IL-1β prevented the differentiation of type II epithelial cells. The production of Clara cell secretory protein in the nonciliated bronchiolar (Clara) cells was likewise suppressed by IL-1β. In conclusion, expression of IL-1β in the epithelium of the distal airways disrupted the development of the airspaces and capillaries in the fetal lung and caused fatal respiratory failure at birth.

Clinical-laboratory and instrument indices of the thyroid gland state after surgical treatment of tumors of the chiasmatic-cellar region

Hypopituitarism is often developed in the result of operative treatment of tumours in chiasmal-cellular area. One of frequent indications of hypophysis’ deficit is decreasing of product of thyroid-stimulating hormone (TSH) that leads to deficit of thyroid hormones. Most patients, who had been operated in terms of tumours of chiasmal-cellular area, have normal volume and structure of thyroid gland in the background of decreased level of free thyroxine and absence of increasing of level of TSH on the principle of feedback, it indicates to the secondary character of destruction of thyroid gland. Patients operated in terms of craniopharyngioma and somatoprolactinoma more often have secondary hypothyroidism. A high per cent of fibrotic changes of tissue of thyroid gland in patients operated in terms of prolactinoma is explain by autoimmune thyroiditis in anamnesis.