Unraveling the Impact of Dab1 Gene Silencing on the Expression of Autophagy Markers in Lung Development.

The purpose of this study was to evaluate the effects of Dab1 gene silencing on the immunoexpression of light chain 3 beta (Lc3b), glucose regulating protein 78 (Grp78), heat shock cognate 71 (Hsc70), mammalian target of rapamycin (mTOR) and lysosomal-associated membrane protein 2A (Lamp2a) in the lung tissue of developing yotari (Dab1-/-) and wild-type (wt) mice. The lung epithelium and mesenchyme of the embryos at gestational days E13.5 and E15.5 were examined using immunofluorescence and semi-quantitative methods. In the pulmonary mesenchyme and epithelium, Grp78 and Lc3b of moderate fluorescence reactivity was demonstrated in wt mice for both evaluated time points, while yotari mice exhibited only epithelial reactivity for the same markers. Mild punctate expression of Hsc70 was observed for both genotypes. A significant difference was present when analyzing mTOR expression, where wt mice showed strong perinuclear staining in the epithelium. According to our data, Dab1 gene silencing may result in autophagy abnormalities, which could then cause respiratory system pathologies via defective lung cell degradation by lysosome-dependent cell elimination.

Disturbances in Switching between Canonical and Non-Canonical Wnt Signaling Characterize Developing and Postnatal Kidneys of Dab1-/- (yotari) Mice.

This study aims to determine the protein expression patterns of acetylated α-tubulin, inversin, dishevelled-1, Wnt5a/b, and ß-catenin in developing (E13.5 and E15.5) and early postnatal (P4 and P14) kidneys of Dab1-/- (yotari) mice, their role in regulating the Wnt signaling pathway, and the possible relation to congenital anomalies of kidney and urinary tract (CAKUT). The analysis of target protein co-expression, observed in the renal vesicles/immature glomeruli, ampullae/collecting ducts, convoluted tubules, metanephric mesenchyme of developing kidneys, but proximal convoluted tubules, distal convoluted tubules and glomeruli of postnatal kidneys, was performed using double immunofluorescence and semi-quantitative methods. The overall expression of acetylated α-tubulin and inversin during normal kidney development increases with higher expression in yotari mice as the kidney acquires mature morphology. An increase in ß-catenin and cytosolic DVL-1 levels, indicating a switch from non-canonical to canonical Wnt signaling, is found in the postnatal kidney of yotari mice. In contrast, healthy mouse kidney expresses inversin and Wnt5a/b in the postnatal period, thus activating non-canonical Wnt signaling. Target protein expression patterns in kidney development and the early postnatal period observed in this study could indicate that switching between canonical and non-canonical Wnt signaling is crucial for normal nephrogenesis, while the defective Dab1 gene product in yotari mice may promote CAKUT due to interfering with this process.

Expression Patterns of Serotonin Receptors 5-HT1A, 5-HT2A, and 5-HT3A during Human Fetal Lung Development.

We analyzed the expression of the serotonin receptors 5-HT1A, 5-HT2A, and 5-HT3A at four different stages of fetal lung development from 12 to 40 weeks of gestation, divided into four groups: the pseudoglandular stage (12-16th week of development; n = 8), the canalicular stage (16th-26th week of development; n = 7), the saccular stage (26th-36th week of development; n = 5), and the alveolar stage (36th-40th week of development; n = 5). The strongest expression of all three receptor types was found in the epithelium of the proximal airways during the pseudoglandular, canalicular, and saccular stages and in a vascular wall. 5-HT1A was also strongly expressed in the smooth muscle cells of the proximal airway. Vascular smooth muscle cells and endothelium occasionally showed a strong expression of 5-HT1A and 5-HT2A. In the alveolar stage, the expression of 5-HT1A, 5-HT2A, and 5-HT3A was detected in both type I (p1) and type II (p2) pneumocytes, with a stronger expression in p2. A significant decrease in percent the 5-HT2A area and in the integrated density was observed at the alveolar stage. On the other hand, a significant decrease in the percentage area but an increase in the integrated density was observed for 5-HT3A toward the alveolar stage, suggesting that a smaller number of cells expressed 5-HT3A but that they (p1 and p2) significantly increased their 5-HT3A expression at the alveolar stage. The results presented provided us with new data on the development and function of the serotonin system in the human fetal lung and gave us insight into their possible involvement in the pathogenesis of lung pathology, particularly that characteristic of the neonatal period.

The Interplay of Cx26, Cx32, Cx37, Cx40, Cx43, Cx45, and Panx1 in Inner-Ear Development of Yotari (dab1-/-) Mice and Humans.

We investigated DAB1-protein deficiency in the inner-ear development of yotari in comparison to humans and wild-type (wt) mice by immunofluorescence for the expression of connexins (Cxs) and the pannexin Panx1. The spatial and temporal dynamics of Cx26, Cx32, Cx37, Cx40, Cx43, Cx45, and Panx1 were determined in the sixth and eighth weeks of human development and at the corresponding mouse embryonic E13.5 and E15.5, in order to examine gap junction intercellular communication (GJIC) and hemichannel formation. The quantification of the area percentage covered by positive signal was performed for the epithelium and mesenchyme of the cochlear and semicircular ducts and is expressed as the mean ± SD. The data were analysed by one-way ANOVA. Almost all of the examined Cxs were significantly decreased in the cochlear and semicircular ducts of yotari compared to wt and humans, except for Cx32, which was significantly higher in yotari. Cx40 dominated in human inner-ear development, while yotari and wt had decreased expression. The Panx1 expression in yotari was significantly lower than that in the wt and human inner ear, except at E13.5 in the mesenchyme of the wt and epithelium and mesenchyme of humans. Our results emphasize the relevance of GJIC during the development of vestibular and cochlear functions, where they can serve as potential therapeutic targets in inner-ear impairments.

Expression and localization of FGFR1, FGFR2 and CTGF during normal human lung development.

Aim of our study was to provide insight into the temporal and spatial expression of FGFR1, FGFR2 and CTGF during normal human lung development which may have an important impact on understanding occurrence of developmental lung anomalies. Morphological parameters were analysed using double immunofluorescence on human embryonal (6th and 7th developmental week-dw) and foetal (8th, 9th and 16th developmental week) human lung samples. FGFR1 and FGFR2 was positive during all the dw in both the epithelium and mesenchyme. The highest number of FGFR1 positive cells was observed during the 6th dw (112/mm2) and 9th dw (87/mm2) in the epithelium compared to the 7th, 8th and 16th dw (Kruskal-Wallis test, p < 0.001, p < 0.0001). The highest number of FGFR1 positive cells in the mesenchyme was observed during the 8th dw (19/mm2) and 16th dw (13/mm2) compared to the 6th, 7th, and 9th dw (Kruskal-Wallis test, p < 0.001, p < 0.0001). The number of FGFR1 positive cells in the epithelium was higher for FGFR2 compared to number of positive cells (Mann-Whitney test, p < 0.0001). FGFR2 showed the highest number in the epithelium during the 7th dw (111/mm2) and 9th dw (87/mm2) compared to 6th, 8th and 16th dw (Kruskal-Wallis test, p < 0.001, p < 0.0001, p < 0.01 respectively). The highest number of FGFR2 positive cells in the mesenchyme was observed during the 9th dw (26/mm2), compared to the 6th, 7th,8th and 16th dw (Kruskal-Wallis test, p < 0.0001), while the number of FGFR2 positive cells in the epithelium was significantly higher than in the mesenchyme (Mann-Whitney test, p < 0.0001). CTGF was negative in both epithelium and mesenchyme during all except the 16th dw in the mesenchyme where it co-localized with FGFR2. FGFR1 and FGFR2 might be essential for epithelial-mesenchymal interactions that determine epithelial branching and mesenchymal growth during early lung development. Sudden increase in FGF1 in the epithelium and FGF2 in the mesenchyme in the foetus at 9th dw could be associated with the onset of foetal breathing movements. CTGF first appear during the foetal lung development.

Scrotal abscess as the first symptom of fatal necrotizing pancreatitis.

Acute necrotizing pancreatitis represents a severe form of acute pancreatitis, characterized by pancreatic collections and necrosis of the pancreas and surrounding tissues. It is associated with higher rates of morbidity and mortality. Scrotal swelling is a rare complication of acute necrotizing pancreatitis due to the extension of pancreatic necrotic masses involving the inguinal canal and scrotum. We report a case of scrotal swelling as the first sign of unrecognized severe necrotizing pancreatitis with a fatal outcome.

Diagnostic Value of Fine Needle Aspiration Cytology for Breast Tumors

Breast cancer is the most frequently diagnosed cancer and the leading cause of death from cancer in women. The accuracy of diagnosis can be increased with a combination of clinical examination, imaging diagnostics, and fi ne needle aspiration cytology (FNAC) or core needle biopsy, also known as triple test. The aim of the study was to evaluate the sensitivity and specificity of FNAC in the diagnosis of breast tumors in our institution by correlating it with histopathology findings. We assessed the accuracy of 124 FNAC findings by comparing cytological diagnosis of breast masses with the diagnoses from histopathology reports obtained by surgery. Statistical analysis showed 95.1% accuracy, 97.7% sensitivity, 89.1% specificity, 95.5% positive predictive value and 94.2% negative predictive value of FNAC. Study results indicated that FNAC could be used as a highly reliable tool in the differential diagnosis of breast tumors, in combination with clinical and imaging findings, especially in developing countries with limited financial resources.

Co-expression of TTF-1 and neuroendocrine markers in the human fetal lung and pulmonary neuroendocrine tumors.

The expression pattern of thyroid transcription factor 1 (TTF-1) and neuroendocrine markers, neuron cell adhesion molecule (NCAM; CD56), chromogranin A (CgA) and synaptophysin (Syp), of different lung cell lineages was histologically analyzed in 15 normal human fetal lungs and 12 neuroendocrine tumors (NETs) using immunohistochemical methods. During pseudoglandular phase strong nuclear TTF-1 staining was detected in the columnar nonciliated epithelial cells, while NCAM, CgA and Syp had a moderate expression in the proximal airways and mild expression in the distal airways. Neuroendocrine cells (NECs) in proximal lung airway were co-localizing TTF-1 and other neuroendocrine markers while neuroendocrine bodies (NEBs) exhibit only staining with NCAM and Syp. In the canalicular phase TTF-1 nuclear staining was expressed only in several epithelial cells in proximal airways, while budding airways epithelium showed strong TTF-1 expression. Expression of NCAM, CgA and Syp in this phase equals the one in pseudoglandular phase. NEBs cells were co-localizing TTF-1 and NCAM in proximal airways and few NECs in distal airway were co-localizing TTF-1 and Syp. TTF-1 staining in the saccular phase was limited to subsets of epithelial cells in the proximal airways with stronger positivity in the distal airways. NCAM expression is moderate only in proximal airways, while Syp and CgA show mild expression in proximal and distal airways. NECs were co-localizing TTF-1 and NCAM in proximal lung airway. With regard to NECs, all small cell lung cancer (SCLC) cells had strong TTF-1, NCAM, Syp and CgA positivity and TTF-1 co-localized with other neuroendocrine markers. All pulmonary typical carcinoids were TTF-1 negative, while pulmonary atypical carcinoids were focal positive for TTF-1 and some neoplastic cells co-localized TTF-1 with neuroendocrine markers. Our results indicate that TTF-1 expression in NECs suggests a possible role in their normal development and differentiation. Our results also indicate that possible cell of origin for poorly differentiated SCLC and some atypical carcinoid could be a progenitor cell in neuroendocrine lineage while in typical carcinoids possible cell of origin is localized in terminally differentiated NECs.

Proliferation, apoptosis and expression of matrix metalloproteinase-9 in human fetal lung.

Expression pattern of the Ki-67, caspase-3 and matrix metalloproteinases-9 (MMP-9) factors were immunohistochemically analyzed in 48 human fetal lungs from 12 to 40 weeks of gestation. The number of Ki-67 positive cells in the epithelium of canaliculare (88cells/mm(2)) and sacculare stage (93cells/mm(2)) were significantly higher than in the epithelium of pseudoglandular stage (12cells/mm(2)) (p=0.0008 vs. p=0.003). The number of Ki-67 positive cells in the mesenchyme of canaliculare stage (132cells/mm(2)) was significantly higher than in the mesenchyme of pseudoglandular stage (37cells/mm(2)) (p=0.001). The proliferation of mesenchymal cells was higher than the epithelial cells in all developmental stages, especially in the canaliculare stage (p=0.007). Similarly, the number of caspase-3 positive cells in the epithelium of canalicular stage (13cells/mm(2)) was significantly higher than in the epithelium of pseudoglandular stage (6cells/mm(2)) (p=0.002) with peaks in the conductive epithelium of canalicular stage. The number of caspase-3 positive cells in the mesenchyme of canaliculare stage (3cells/mm(2)) was significantly higher than in the mesenchyme of saccular stage (0cells/mm(2)) (p=0.05). There were no caspase-3 positive cells in the mesenchyme of pseudoglandular stage. However, unlike the Ki-67 expression, mesenchymal cells in comparison to epithelial cells express substantially less caspase-3 in all developmental stages. Up to the saccular stage, the expression of MMP-9 in mesenchymal cells showed a linear increase with most pronounced expression in that stage. The number of MMP-9 positive cells in the mesenchyme of canaliculare (20cells/mm(2)) and sacculare (39cells/mm(2)) stage were significantly higher than in the mesenchyme of pseudoglandular stage (12cells/mm(2)) (p=0.04 vs. p=0.004). The first epithelial cells that express MMP-9 were present only at the alveolar stage. Increased proliferation and apoptosis of the mesenchymal cells of canalicular stage is important for formation of definite structures within the stroma of the lung parenchyma. Although apoptosis in the epithelium is not pronounced as proliferation, it is important for thinning of the epithelium and consequent spread of respiratory tract. However in the saccular stage when mesenchyme disappears, MMP-9 expression is more important for primitive alveoli differentiation.