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Activation of the IL-6 mediated JAK-STAT (Janus associated kinase-signal transducer and activator of transcription) oncogenic signalling plays a major role in hepatocellular carcinoma pathogenesis. The aim of this study is to assess the anti-tumour, anti-proliferative and apoptotic potential of artesunate and its capacity to modulate JAK-STAT pathway in a nitrosodiethylamine mediated experimental hepatocellular carcinoma model. Administration of nitrosodiethylamine (200 mg/kg body weight by i.p. Injections) to rats resulted in alterations of liver pathophysiological parameters such as increased relative liver weight, and increased tumour nodule occurrence. It also increased the levels of serum marker enzymes (AST, ALT, ALP, LDH, and γGT) and tumour biomarker (AFP) levels suggestive of its capacity to cause liver tumourigenesis. Additionally, the immunohistochemistry of liver sections pertaining to nitrosodiethylamine administered animals showed increased detection of AgNOR, PCNA, and GST-Pi positive cells suggestive of its capacity to promote liver proliferation associated tumourigenesis. On the contrary, artesunate (25 mg/kg bodyweight) supplementation to nitrosodiethylamine administered animals decreased all the above mentioned pathophysiological, biochemical, and immunohistochemistry parameters suggesting its anti-tumour and anti-proliferative potential. Furthermore, immunoblot analysis showed significant up-regulation of IL-6, GP130, JAK-2, STAT-3 (pY705), Bcl-xL, Bcl-2 and simultaneous down-regulation of Caspase-3, PARP and SOCS-3 in nitrosodiethylamine administered animals. Nevertheless, the immunoblot analysis revealed vice-versa on artesunate supplementation to nitrosodiethylamine administered animals, indicating promotion of the feedback loop inhibition mechanism through SOCS3 up-regulation thereby leading to suppression of JAK-STAT signalling. Overall all these findings substantiate that artesunate promotes anti-tumour, anti-proliferation and apoptosis against nitrosodiethylamine mediated hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Curcumin has been reported to suppress different types of clinical and experimentally-induced tumors, but due to less absorption and quick metabolism it show poor bioavailability. The present study was envisaged to investigate the possible synergistic effect of combined treatment of curcumin with piperine in suppression of diethylnitrosamine (DENA)-induced hepatocellular carcinoma (HCC) in rats, owing to permeability enhancing effect of latter. HCC was induced by supplying DENA (0.01%) in drinking water for 10 weeks. The rats were treated with curcumin (100 mg/kg; p.o. ) per se and curcumin along with piperine (20 mg/kg; p.o. ) for 4 weeks post HCC induction. The combined treatment significantly attenuated the morphological, histopathological, biochemical, apoptotic and proliferative changes in the liver and serum in comparison to curcumin per se and vehicle control group. The results of present study concluded that curcumin in combination with piperine shows better suppression of DENA-induced HCC in contrast to curcumin per se . [ABSTRACT FROM AUTHOR]

The effect of silibinin on lipid profile, colonic mucin content, transforming growth factor beta (TGF-β) expression, cell proliferation and apoptosis in high fat diet fed rats challenged with 1, 2 dimethyl hydrazine (DMH) was investigated. Rats were randomized into six groups. Group 1: Control rats; Group 2: Silibinin (50 mg/kg of b.w. p.o every day); Group 3: DMH (20 mg/kg b.w.); Groups 4–6: DMH + Silibinin (Initiation, Post-initiation and entire period treatment regimens, respectively). At the end of 32nd week, DMH alone administered rats showed hyperlipidaemia and colon tumour burden with decreased mucin content. The expression of TGF-β was found to be lost in the early adenoma but increased stromal positivity of TGF-β was observed in adenocarcinoma of the colon. Evasion of apoptosis is also seen in the colon of tumour bearing rats. These DMH induced pathological changes were attenuated by silibinin supplementation reflecting its antihyperlipidaemic, colon protective and anticarcinogenic effects. [ABSTRACT FROM AUTHOR]

The salubrious effects of 3-Formylchromone (3-FC) against nitrosodiethylamine (NDEA) mediated early hepatocellular carcinogenesis was investigated in vivo by this study. Male Wistar rats were administered three interspersed intraperitoneal injections of NDEA (200 mg/kg body weight) until sixth week, followed by, thrice a week oral dose of 3-FC (25 mg/kg body weight) from the seventh week to eleventh week. Oral supplementation of Wistar rats with 3-FC prevented the increase in serum marker enzymes (AST, ALT, LDH) and serum pre-neoplastic marker (γ-GT) induced by NDEA. Biochemical observations were found to be further correlated with histological studies, indicating the potential of 3-FC to mediate suppression of hepatic damage/pre-neoplastic lesions. Argyrophilic nucleolar organizer region (AgNOR) staining was done in histology sections to confirm the anti-proliferative potential of 3-FC against NDEA-induced early hepatocellular carcinogenesis. RT-PCR and immunoblot analysis was done to find the modulations in the gene transcript/protein level expression of pre-neoplastic marker (GST-pi), proliferation marker (PCNA), apoptotic mediators (PPARγ, NFκB-p65 and p53). 3-FC was found to favorably modulate the expressions of GST-pi, PCNA, PPARγ, NFκB-p65, p53 clearly confirming the anti-proliferative and apoptotic potential of 3-FC. Further, the apoptotic effect of 3-FC against NDEA-induced early hepatocellular carcinogenesis was confirmed by caspase-3 activity assay and DNA fragmentation analysis. Based on these findings, it is concluded that 3-FC possesses hepatoprotective, anti-pre-neoplastic, anti-proliferative and apoptosis inducing capability against NDEA-induced early hepatocellular carcinogenesis. [ABSTRACT FROM AUTHOR]

Abstract: The effect of glue snuffle on the expression of argyrophilic nucleolar organizer regions (AgNORs), an indicator of ribosome biosynthesis, in epithelial cells of oral mucosa has been investigated. AgNOR was evaluated by cytochemical staining in 148 Indian street boys (median age 12 year) who had different bad addictions like tobacco smoking, chewing and most importantly inhaling glue and 20 age- and body mass index-matched school boys who had no such type of bad habit. Compared with school boys, glue addicted street boys showed remarkably increased number of AgNOR dots per nucleus (9.38±1.84 vs. 3.12±0.87, p <0.001), AgNOR size (1.34±0.52 vs. 0.43±0.02μm2, p <0.001) and percentage of AgNOR occupied nuclear area (9.38±2.12 vs. 0.99±0.03%, p <0.001). Increase in number and size of the dots is also higher in tobacco smokers and chewers when compared with school boys but a remarkable difference was recorded in glue addicted boys. The changes in AgNOR expression were positively associated with years of addiction after controlling potential confounders. Thus, glue snuffle appeared to be a risk factor for abnormal cell growth via up-regulation of ribosome biogenesis. [Copyright &y& Elsevier]

Abstract: This study presents the development and evaluation of an image processing software for computer-assisted cellular structure counting. The proposed software consists of a set of processing and analytical tools which allows its use in several applications of cell and cellular structure counting. A particular application in AgNOR quantitative analysis is presented. The knowledge obtained from experienced pathologists has been codified in a sequence of processing steps in order to allow automatic estimation of the mean number of AgNORs per cell in ameloblastomas. The performance of the presented software in such application was verified by comparing the data provided by visual analysis, by two observers previously calibrated and under supervision of two experienced specialists (Group 1) and by the computer program (Group 2). No statistical difference was observed (p <0.05) between the two groups. The use of the proposed method in AgNOR applications permitted attainment of accurate and precise data without the difficulties frequently found in the traditional visual analysis method (time, training and subjectivity). The developed software is an interesting tool as an aid in the study (estimation of the number of cells and cellular structures) of malignant and benign neoplasms. [Copyright &y& Elsevier]

Abstract: The involvement of the main pallial subdivisions of the teleost telencephalic pallium in spatial cognition was evaluated in a series of three experiments. The first two compared the effects of lesions selective to the lateral (LP), medial (MP) and dorsal (DP) telencephalic pallium of goldfish, on the retention and the reversal learning of a spatial constancy task which requires the use of allocentric or relational strategies. The results showed that LP lesions produced a selective impairment on the capability of goldfish to solve the spatial task previously learned and on the reversal learning of the same procedure, whereas MP and DP lesions did not produce observable deficits. The third experiment evaluated, by means of the AgNOR stain, learning-dependent changes of the neuronal transcription activity in the pallium of goldfish trained in the spatial constancy task or in a cue version of the same procedure, which only differed on their spatial cognition demands. The results revealed that training in the spatial task produced an increment in the transcriptive activity which was selective to the neurons of the ventral lateral pallium, as indicated by increases in the size of the nucleolar organizing region (NOR), the nucleolar organelles associated with the synthesis of ribosomal proteins. In contrast, training in the cue version did not produced observable changes. These data, revealing a striking functional similarity between the lateral telencephalic pallium of the teleost fish and the amniote hippocampus, provide additional evidence regarding the homology of both structures. [Copyright &y& Elsevier]

Abstract: Down''s syndrome (DS) or trisomy 21 is the most frequent genetic birth defect associated with mental retardation. Although DS has been known for more than a 100 years and its chromosomal basis recognized for half a century (1959), the underlying patho-mechanisms for the phenotype formation remain elusive and cannot be fully explained by simple gene dosage effect. The general consensus is that the extra chromosome 21 genes perturb the global metabolism of the body cells. Our experiments show that the most prominent metabolic perturbation occurs during ribosome biogenesis in the cells of DS babies/infants. In humans, ribosomal RNA (rRNA) gene families or nucleolar organizer regions (NORs) are localized at the secondary constriction (on the satellite stalks) of five pairs of acrocentric chromosomes (13, 14, 15, 21 and 22) and their activities are evaluated specifically either in metaphase or interphase through a procedure known as AgNOR or silver staining. Our successive AgNOR studies, supported by RNA and nuclear protein measurement, show that cells from DS infants produce more ribosomes than expected, accounting for the extra set of active rRNA gene family (1/6–1/11) situated on the extra chromosome 21. Thus, the presence of an extra chromosome 21 stimulates a global increase in ribosome biogenesis in cooperation with other NOR-bearing chromosomes, causing unnecessary rRNA and ribosomal proteins synthesis compared to controls. Following the description of NORs, AgNOR, AgNOR-proteins, AgNOR measurement and our experimental results, we propose that the extra RNA and protein synthesis can cause a fundamental handicap to DS infants, contributing to the formation of DS phenotypes, due to the wasted energy in producing unnecessary macromolecules, including energy (GTP)-dependent transport of the excessive ribosomes from the nucleus to the cytoplasm. [Copyright &y& Elsevier]

Abstract: Background: Patients with schizophrenia show a significantly higher frequency of hyperbilirubinemia than patients suffering from other psychiatric disorders and the general healthy population. We examined the hyperbilirubinemia on behavioral and neuropathological changes in rats as a possible animal model of schizophrenia. Methods: Gunn rats with severe hyperbilirubinemia (j/j), Gunn rats without severe hyperbilirubinemia (+/j), and Wistar rats were examined by open-field, social interaction, and prepulse inhibition tests. TUNEL, AgNOR and Ki-67 were also assayed on paraffin-embedded brain sections of these rats. Results: Compared to Wistar rats, both Gunn j/j and +/j rats showed hyperlocomotion, high sniffing scores, and low defecation scores. They showed significantly more aggressive behaviors and impaired prepulse inhibition. The numbers of Ki-67-labeled cells and AgNOR were lower and the number of TUNEL-positive cells was higher than that of Wistar rats. Conclusions: These results might support the neurodevelopmental hypothesis of schizophrenia. Both Gunn j/j and +/j rats may be a useful animal model and provide clues to the role of hyperbilirubinemia in schizophrenia. [Copyright &y& Elsevier]

[Display omitted] • Only Leishmania -infected dogs showed biochemical alterations. • AgNORs per cell were significantly higher in clinically affected dogs. • Enhanced histopathological lesion score was observed in Leishmania -infected dogs. • Number of NORs and histopathological lesion score was positively correlated. • AgNOR expression and renal lesions were associated with clinical signs. Hyperactivation of tubular cells contributes for the progression of kidney lesions. The exacerbated expression of immunological proteins and ribosomal DNA (rDNA) transcriptional activity are observed in tubular cells. This intensified expression results in more prominent hypertrophic changes and is often accompanied by increased expression of factors involved in different phases of ribosomal biosynthesis, such as the nucleolar organizer regions (NOR). The aim of this study was to evaluate whether there is an association between NOR proteins, renal impairment, and clinical status in Leishmania -infected dogs (CanL). Forty-five dogs with CanL and six uninfected controls were assessed in this study. PCR was performed to detect parasites' nucleic acids in kidney. Histopathological analyses were performed in kidney fragments, and NOR was detected by Ag stain (AgNOR). Leishmania -infected dogs showed more intense inflammation and collagen deposition compared with uninfected controls. Biochemical alterations were observed only in Leishmania -infected dogs. AgNORs per cell were significantly higher in clinically affected dogs and higher histopathological lesion score was observed in Leishmania -infected dogs. Positive correlations between number of NORs per cell in medullary region and histopathological lesion score were observed. Furthermore, AgNOR expression, intensity of renal lesions, and clinical sigs was associated in Leishmania -infected dogs. We propose that the detection of AgNOR proteins could be used to better estimate the kidney tubular damage at the time of examination in Leishmania -infected dogs as a marker to estimate renal impairment in dogs with CanL. [ABSTRACT FROM AUTHOR]

Summary: The argyrophylic staining of the nucleolar organizer regions (AgNOR positive response) in interphase nuclei is often related directly to the cellular demand for ribosome biogenesis and is considered of relevance in studies of tumor pathology. Transformation of human breast epithelial MCF-10A cells by the c-Ha-ras oncogene results in altered growth, invasiveness and tumorigenicity in nude mice. Since ras transformation may be associated with a more intense nucleolar activity, we examined the influence of transfection by the Ha-ras oncogene on AgNOR staining response in MCF-10A cells. Following assessment of the AgNOR response with video image analysis, the AgNOR-positive areas and the AgNOR area/nuclear area ratio, but not the number of AgNOR aggregates or dots per nucleus, were found to be much higher after ras transformation. A role of the Ha-ras transformation on the nucleolar activity of the MCF-10A is thus suggested as assessed by the AgNOR staining. Based on data in the literature, it is also hypothesized that a decreased wild-type p53 level, possibly promoted by the ras transformation, may be associated with the increased AgNOR response. [Copyright &y& Elsevier]

Summary: Abnormalities in any component of the cell cycle regulatory machine may result in oral cancer, and markers of cell proliferation have been used to determine the prognosis of tumor progression. The aim of this study was to determine whether silver-stained nucleolar organizer region (AgNOR) and Ki-67 measurements could improve the assessment of growth rates in oral lesions. Eighty-three oral biopsies were studied, 20 of which were classified as fibrous inflammatory hyperplasia (FIH), 40 as leukoplakia (LKP) and 23 as oral squamous cell carcinoma (OSCC). Within the LKP group, 22 out of 29 biopsies were diagnosed as non-dysplastic leukoplakia (LK) and 18 as dysplastic leukoplakia (DLK), presenting discrete, moderate and severe dysplasia. Ki-67 immunolabeling of the lesions increased steadily in the following order: FIH, DLK, LK and OSCC, indicating that Ki-67 is a good marker for predicting the proliferative fraction among benign, premalignant and malignant oral lesions. The median values of AgNOR parameters indicate that the morphometric index gives better results regarding the proliferative rate than the numerical one. A series of linear regressions between AgNOR parameters and Ki-67 showed positive associations. We conclude that a combination of Ki-67 and morphometric AgNOR analyses could be used as an aid in the determination of the proliferative status of oral epithelial cells in oral cancer. [Copyright &y& Elsevier]

Abstract: Traditional criterions are not sufficient to predict accurately the recurrence of transitional cell carcinoma of the urinary bladder. Therefore, we aimed to evaluate the AgNORs via total AgNOR area/nucleus area (TAA/NA) for each cell as a prognostic parameter, in TCC of urinary bladder. Tumor tissues of 20 consecutive cases of male bladder cancer patients were divided into two groups as middle differentiated (LG) and high grade (HG). The extra-tumoral tissue (ETT) samples of 10 males served as control group. A second control group (HC) consisted of five healthy and normal bladder tissue samples. The 3μm of sections from each paraffin embedded tumoral, extra-tumoral and normal tissue samples served as patient and control groups. After deparaffinization and rehydratation steps, silver (AgNO3) staining of nucleolar organizer regions-associated proteins (AgNORs) was performed. Instead of Giemsa stain, we used Hematoxylin for contra staining. The images of the 100 analyzable nuclei from each tissue sample, transferred by means of a video camera and video capture card from microscope and recorded onto a computer. Software was prepared in Delphi language for analysis. Mean (E+02) TAA/NA values of HC, ETT, LG and HG groups were 6.97+2.80, 5.70+1.82, 7.80+3.22 and 9.24+3.88, respectively. Statistical comparisons have shown significant differences between all groups.In conclusion, mean TAA/NA per cell has a potential to be a prognostic parameter. Therefore, further evaluation of big patient series will be useful. [Copyright &y& Elsevier]

Abstract: The purpose of the current study was to evaluate the effect of alcohol on the proliferative activity of epithelial cells in the lingual mucosa of mice by means of silver-staining nucleolar organizer region (AgNOR) count and area measurements. Forty-eight CF1 mice were separated into three groups. The test groups were submitted to topical exposure to, or intake of, 40% (volume/volume) ethyl alcohol. Biopsy specimens were collected from the middle third of the dorsal tongue at 0, 6, and 12 months, and samples were stained according to the AgNOR technique. Mean number and mean area of AgNOR per nucleus were calculated for 50 basal layer cells and 50 intermediate layer cells. Increases in mean number and mean area of AgNOR per nucleus in intermediate cells were observed at 12 months in the alcohol intake group (P &#60; .05). Results showed that intake of 40% alcohol increased epithelial cell proliferation in the dorsal surface of lingual mucosa. [Copyright &y& Elsevier]

Nucleolar organizer regions (NOR) are chromosomal segments in which ribosomal RNA is encoded. AgNOR technique, which reveals these regions, has rarely been used in the diagnosis and preceding the prognosis of pituitary adenoma. The purpose of this study was to determine and evaluate the correlation of hormonal profile with AgNOR counts, the correlation of recurrence with AgNOR counts, and to determine the consistency between the evaluations of the two independent observers. This study covers 33 patients with pituitary adenoma. The slides were stained with hematoxylin and eosin. Silver colloid method was used for NOR, and immunohistochemistry for hormone expressions. Consistency was significant between the two pathologists (for hormonal profile, the Kappa test was used and P<0.001; for AgNOR count, intra-class correlation coefficient was 0.83). There was no significant correlation between mean AgNOR counts and hormone expressions (P>0.05), and there was also no correlation between mean AgNOR counts and tumor sizes (P>0.05). While no difference between recurrent and non-recurrent patients was found with respect to mean AgNOR numbers (P>0.05), nucleolar silver staining in the form of AgNOR clusters was more frequently detected in recurrent cases. [Copyright &y& Elsevier]

The aim of the present study was to examine age-related changes in the proliferative capacity of acinar and ductal cells in labial salivary glands of healthy subjects as reflected by AgNOR and Ki-67 parameters. The study was conducted on 24 postmortem samples of labial salivary glands free from salivary gland tumors/diseases. Samples were equally divided into three age groups: young (mean age 17 yr), adult (mean age 38 yr) and old (mean age 84 yr). The number of profiles of AgNOR (nNOR) and AgNOR profile area per nucleus (TVNOR) were histomorphometrically assessed by CUE-3 automatic image analyzer on 30 nuclei of acinar and intercalated ductal cells in each age group. The percentage of Ki-67 positively stained acinar and ductal cells was evaluated histomorphometrically. Statistical analysis was carried out by one-way ANOVA and Kruskal–Wallis tests. In the acinar cells, nNOR increased significantly with aging (p<0.00001), while TVNOR did not change significantly. In the ductal cells, both nNOR and TVNOR increased significantly (p<0.00001) with a relatively higher increase in TVNOR. Since only isolated acinar and ductal cells demonstrating Ki-67 positive nuclei were found in the different age groups, the overall proliferation index was estimated to be 0%. Thus, it is suggested that age-related changes in AgNOR parameters could reflect changes in the metabolic cell activity rather than changes in their proliferative capacity. In this context, AgNOR results are in accordance with the histomorphometric and physiologic age-related changes occurring in labial salivary glands. [Copyright &y& Elsevier]