[Role of the Nucleolus in Rearrangements of the IGH Locus].

The review summarizes the results from a series of studies focusing on the role that the nucleolus plays in maturation of the IGH locus and the choice of its partner genes in leukemia-associated translocations. The role of nuclear compartmentalization and nuclear localization of translocated oncogenes in ectopic activation of their transcription is discussed.

The value and significance of nucleolar organizer region proteins as markers of malignancy in breast cancer patients.

OBJECTIVES: To assess argyrophilic nucleolar organizer regions (AgNORs) in 60 patients with primary breast carcinoma and evaluated their association with clinical prognostic parameters of breast cancer. METHODS: Argyrophilic nucleolar organizer regions were stained in paraffin sections of the tissues using Ploton's silver method. For each sample, the number of AgNORs within the nuclei of 100 tumor cells was counted. The average number of AgNORs per nucleus was calculated, and the results were expressed as mean. RESULTS: The number of AgNORs was significantly higher in breast invasive ductal carcinoma (6.6) compared to benign breast tumors (fewer than 2.0). However, differences in AgNOR counts across different age groups were not statistically significant. CONCLUSION: This study suggests that AgNOR counts could be used as a potential procedure for estimating proliferation characteristics in histopathological sections of benign and malignant breast lesions. Argyrophilic nucleolar organizer region counts may also be valuable for identifying high-risk patients and indicating tumor aggressiveness. A larger study with an increased sample size that incorporates both AgNOR numbers and Ki67 scores for assessing cell kinetics is needed to confirm our findings.

Antiproliferative efficacy of hesperetin (citrus flavanoid) in 1,2-dimethylhydrazine-induced colon cancer.

Cancer is the second leading cause of death worldwide and is increasing at an alarming rate. The present study was to evaluate the antiproliferative effects of hesperetin, a flavonoid commonly found in many herbal medicines and foods, on aberrant crypt foci (ACF), argyrophylic nucleolar organizer regions (AgNORs) and proliferating cell nuclear antigen (PCNA) in 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats. Rats were given subcutaneous injections of DMH (20 mg/kg body weight) weekly for 15 weeks to induce carcinogenesis, and hesperetin was administered orally at the dose of 20 mg/kg body weight. DMH exposure alone produced a high incidence of ACF and showed positive staining for PCNA and AgNORs in colonic tissues. Supplementation with hesperetin lowered the PCNA labeling index and suppressed the formation of ACF in the rats with colon cancer. These results clearly reveal that dietary hesperetin possesses antiproliferative ability against chemically induced colon tumourigenesis.

Exfoliative cytological assessment of apparently normal buccal mucosa among quid chewers using argyrophilic nucleolar organizer region counts and Papanicolaou staining.

OBJECTIVE: Quid chewing is associated with an increased risk of oral cancer. This study aims to analyze argyrophilic nucleolar organizer region (AgNOR) counts along with Papanicolaou (PAP) staining in exfoliative smears of quid chewers and non-chewers to correlate quid chewing habits with possible early cytological changes in apparently normal buccal mucosa. STUDY DESIGN: Exfoliative smears were obtained from normal buccal mucosa of 30 male quid chewers and non-chewers. The smears were stained using the AgNOR silver staining technique to evaluate the proliferative activity and PAP for cytological appearance. RESULTS: Statistically higher AgNOR counts were observed in chewers as compared to non-chewers. The difference in the mean percentage of nuclei having ≥5 AgNORs in both groups was statistically significant (p = 0.001). In chewers, PAP showed 77% with class I and the remaining 23% were class II, while the non-chewers showed only class I cytology. AgNOR counts between chewers and non-chewers having class I cytological appearance demonstrated a greater mean AgNOR count in chewers (p = 0.0001). CONCLUSION: Quid chewing seems to have a definite role in promoting proliferative activity of apparently normal buccal mucosal cells. Exfoliative cytological assessment of a combination of AgNOR counts and PAP has the potential for prediction of early quid-associated cellular changes before the appearance of clinical premalignant and malignant lesions.

Evaluation of proliferation activity in dysplastic and nondysplastic oral lichen planus through the analysis of argyrophilic nucleolar organizer regions.

Lichen planus is a mucocutaneous disease, and its potential for malignant transformation is a subject of controversy. We found dysplastic changes in slide review of about 11% of oral lichen planus (OLP) in our previous research. Dysplastic changes can be an initial phase for carcinogenesis. The question is whether it is possible to detect early malignant changes in OLP through the evaluation of proliferation activity. The aim of this study was to compare the cell proliferation activity in dysplastic and nondysplastic oral lichen planus using the quantitative and qualitative analysis of argyrophilic nucleolar organizer region (AgNOR) and to compare candidal infection in these 2 groups. In this cross-sectional, observational, and analytical study, all 14 cases of confirmed dysplastic OLPs and 17 nondysplastic OLPs were stained with an AgNOR technique. Analysis of argyrophilic nucleolar organizer region dots were counted in 100 cells of basal and parabasal layers, and proliferation index (cells with ≥ 5 dots in nucleus) and variations in size of AgNOR dots were also evaluated. Periodic acid-Schiff staining was performed for detection of candidal infection. Mean AgNOR count in dysplastic OLP (2.39 ± 0.2) was more than nondysplastic OLP (2.27 ± 0.59). Candidiasis was found in 14.3% of dysplastic OLP and in 12.5% of nondysplastic OLP. There was no statistically significant difference between the 2 groups in mean AgNOR count (P = 0.62), variation in size of AgNOR dots (P = 1), proliferation index (P = 0.53), and candidiasis (P = 1). The current study showed slight difference in proliferation rate and candidal infection between dysplastic and nondysplastic OLP.

[Suicide and depression in the postmortem evaluation of disturbed neuronal activity]. / Samobójstwo a depresja w swietle posmiertnej oceny zaburzen aktywnosci neuronalnej.

The authors have reviewed the results of their postmortem neurohistological investigations related to the evaluation of impaired activity of brain structures relevant to pathophysiology of depression. Chronic changes in ribosomal DNA transcriptional activity have been indirectly suggested by the AgNOR staining method in neurons of the prefrontal limbic regions, the amygdala, the external globus pallidus, and the serotonergic dorsal raphe nucleus. The impact of processes leading to suicide has been clearly accentuated in contrast to the weak influence of unipolar-bipolar dichotomy. The present results support the view that suicide and depression constitute pathological phenomena with partially different neurobiology. They may be also relevant to forensic pathology.

Adapted technique for demonstrating argyrophilic nucleolar organizer regions in the cytologic samples of canine mast cell tumors.

INTRODUCTION: Argyrophilic nucleolar organizer regions (AgNORs) are nonhistone argyrophilic nucleolar proteins associated with ribosomal genes found in the nucleolar organizer region that reflect cell proliferation and have an affinity for silver. AgNOR staining may be useful to evaluate prognosis in several neoplasms because higher AgNOR counts are related to higher grade tumors, metastases, and shorter survival times. OBJECTIVE: We aimed to report on a quick and practical technique to identify AgNORs adapted for use in routine cytology. MATERIALS AND METHODS: The cytopathologic diagnosis of mast cell tumor (MCT) in samples collected by fine-needle aspiration (FNA) was determined. Next, slides were impregnated with a solution containing silver nitrate; the main modification of our technique included incubation of these slides at a controlled temperature of 25 °C. Some slides were previously stained with Diff-Quik and others were only fixed with methanol. The slides were analyzed under a microscope, and the number of blackened intranuclear points (AgNORs) was counted. RESULTS: Slides prestained with Diff-Quik were easily counted compared with slides only fixed in methanol. Technical issues encountered with the methanol-fixed slides included insufficient cellularity, background precipitation, and an absence of silver impregnation. CONCLUSIONS: The technique reported in this study showed satisfactory results for AgNOR counting in cytologic smears from MCT, such as good impregnation and the elimination of background interferents. Further evaluation of this method comparing AgNOR counts with histologic examinations, tumor grades, other prognostic markers, and survival times are needed to fully evaluate the benefit of this technique.

Cytological Screening Model of Normal Oral Mucosa Exposed to Carcinogens: A Pilot Study.

INTRODUCTION: Oral cytopathology is able to detect incipient cellular alterations, but it is not routinely applied to this purpose. We aimed to establish a model to screen individuals with no oral lesion exposed to smoking/alcohol, by means of the nuclear area, cell proliferation rate, and analysis of genetic damage. METHODS: In this cross-sectional pilot study, 90 patients were allocated into 3 groups: oral cancer group (patients with oral squamous cell carcinoma), tobacco/alcohol group (patients without oral lesions and exposed to these risk factors), and control group (individuals with no lesion and not exposed to tobacco and alcohol). The cytological smears performed in these individuals were stained with Papanicolaou, a silver-staining and a Feulgen reaction. The nuclei of cells were measured, and AgNORs/nucleus and micronuclei (MN) were quantified. The cutoff values were stipulated evaluating the healthy mucosa (control group) and the cancerization field mucosa (oral cancer group). RESULTS: Cutoff values for the screening of individuals exposed to carcinogens were ≥8% of nuclei larger than 100 µm2, ≥3.38 AgNOR/nucleus, and ≥3 MN per 1,000 cells. CONCLUSIONS: Nuclear area measurement and AgNORs/nucleus and MN quantification identified the incipient phase of oral carcinogenesis. A screening model for individuals without oral lesion exposed to smoking/alcohol was proposed.

Correlation between single photon emission computed tomography, AgNOR count, and histomorphologic features in patients with active mandibular condylar hyperplasia.

PURPOSE: To analyze the association between nuclear medicine examination (single photon emission computed tomography [SPECT]), histology, and Argyrophilic Nuclear Organizer Region (AgNOR) count in patients with active condylar hyperplasia who have undergone condylectomy. PATIENTS AND METHODS: Eight patients with a diagnosis of active condylar hyperplasia and evidence of facial asymmetry, with progressive deformation in time and on SPECT studies, were evaluated. The relationship between the rate of technetium Tc 99 intake, cartilage layer thickness, and cellular activity measured by recounting nucleolar organizers with AgNOR was evaluated. RESULTS: The 4 pathologic layers of condylar hyperplasia (fibrous, mesenchymal, and hypertrophic chondrocyte layers and ossification layer) showed great variability and different thicknesses among the cases analyzed. As age increased, the histologic layer thickness decreased (r = -0.73, P = .04). The age of the patients was inversely related to the number of AgNOR dots (r = -0.65, P = .08). The thickness of both mesenchymal and hypertrophic chondrocyte layers was related to cartilage island depth (r = 0.81, P = .02). CONCLUSIONS: Younger patients with condylar hyperplasia had a thicker condylar layer and more cellular activity measured by AgNOR count. The histologic features of this group of patients could not be associated with their SPECT findings.

Correlation between argyrophilic nucleolar organizer region staining and brain tumor classification and grading.

Distinctions between benign and malignant tumors are less evident in the central nervous system than in other tissues. Since the level of cell proliferation is an important feature in tumor grading, we applied AgNOR in 50 cases of brain tumors with different grades and histological origins in order to check its efficiency in discriminating between benign and malignant cases. We found significant differences between the means of grade I (1.76) and grade IV (2.46) tumors. No significant differences were observed considering the same grading with distinct histological types or age of patients, reinforcing the efficiency of AgNOR.

Quantitative analysis of argyrophilic nuclear organizer regions in giant cell lesions of jaws.

BACKGROUND: Giant cell lesions of the jaws are considerably similar according to histopathologic characteristics yet show different clinical behaviors. These lesions include central giant cell granuloma (CGCG), aneurysmal bone cyst, Cherubism, and Brown tumor associated with hyperparathyroidism. The present study aimed to investigate AgNORs count in these lesions as a proliferative marker and to determine whether it can be used to discriminate between them or not. METHODS: Forty-one cases of giant cell lesions of jaws were retrived from Oral Pathology Department (1987-2007). They included 21 cases of CGCG, eight cases of aneurysmal bone cyst (ABC), six cases of Cherubism, six cases of Brown tumor. The mean AgNORs count was calculated for all cases. To compare mean AgNORs in groups of lesions, ANOVA test was performed. RESULTS: Mean AgNOR counts were: (0/85 +/- 0/29) in CGCG, (0/76 +/- 0/32) in ABC (0/87 +/- 0/10) in Cherubism and (0/82 +/- 0/16) in Brown tumor. A significant difference was not observed in AgNOR counts among these groups of lesions. CONCLUSIONS: Jaws giant cell containing lesions have no acceptable differences in mean AgNORs.

Argyrophilic nucleolar organiser region (AgNOR) study of some odontogenic cyst epithelium.

AIMS AND OBJECTIVES: This study compared the biologic activity of three different cyst types and assessed the value of the AgNOR technique as an aid to diagnosis. BACKGROUND: The nucleolar organiser region (NORs) reflect replicatory activities within the cells. It has been found to be of diagnostic value in the prediction of the biologic behaviour of some tumors and even oral epithelial dysplasia. MATERIALS AND METHODS: Thirty histologically diagnosed H & E sections of cystic lesions consisting of ten (10) radicular, ten (10 dentigerous and ten (10) odontogenic kerotocysts were randomly selected from the record of biopsy services of the Lagos University Teaching Hospital. The paraffin blocks of the selected sections were retrieved for AgNOR staining. For each cyst type the mean and standard deviation of AgNORs/100 cells was calculated and compared with other cyst types using an analysis of variance and student's t-tests. RESULTS: RESULTS of this study show a statistically significant higher means AgNORs cyst. (p = 0.0016; p = 0.00049 and respectively). However, no statistically significant difference in mean AgNOR score/cell was found when the radicular and dentigerous cysts were compared. CONCLUSION: These variations reflect the difference in the proliferation rate of the odontogenic keratocyst, radicular and dentigerous cysts.

Oral cytology revisited.

During the last decade, oral cytology has once again become the focus of scientific research. This new interest is due to the introduction of a cytobrush for cell collection as well as a computer-assisted analysis (Oral CDx). Although promising, the sensitivity and specificity of conventional oral brush cytology remains limited. To circumvent the problems and improve the accuracy, various adjunctive analytical methods have been attempted. DNA analysis, immunocytochemical and molecular analysis are suggested methodological cytology approaches to improve the validity of oral brush cytology. An increase in sensitivity (up to 100%) and specificity (up to 100%) of oral brush biopsy has been reported on localized pre-malignant and malignant lesions. Oral brush biopsy probably will not replace histopathology in the definitive diagnosis of oral cancer, but it might be valuable for the prevention of misdiagnosis of clinically doubtful oral lesions and for the monitoring of lesions that might proceed on to oral cancer.

Analysis of cell cycle phases and proliferative capacity in mice bearing melanoma maintained on different dietary proteins.

BACKGROUND: Diet seems to represent, directly or indirectly, 35% of all cancer reports. In this study, the influence of dietary protein on the growth of melanoma B16F10 was evaluated through analyses of cell cycle phases and proliferative capacity. METHODS: Flow cytometry and argyrophilic nucleolar organizer regions (AgNORs) technique were applied in mice bearing B16F10 melanoma cells fed on different dietary proteins. All data were submitted to statistical analyses. RESULTS: The G0/G1 phase increased for the animal groups fed bovine collagen hydrolysate (BCH) or BCH-P1 + whey protein isolate (WPI), compared with mice receiving only WPI, for all dietary groups treated and nontreated with paclitaxel. Mice that received BCH + WPI treated with paclitaxel showed the highest percentage of apoptosis compared with WPI group. AgNORs, total nucleolar organizer regions (NORs)/cells and dot number/cell for all dietary protein groups nontreated with paclitaxel were higher than for the WPI. The only two dietary protein groups treated with paclitaxel that presented higher total NORs and dot number/cell than the WPI group were BCH + WPI and BCH-P1 + WPI. CONCLUSIONS: A significantly lower proliferative capacity and larger number of cells in the G0/G1 phase were observed for the dietary protein groups combining the two collagen hydrolysates, BCH or BCH-P1 with WPI, treated with paclitaxel.

Quantitative estimation of AgNORs in inflammatory gingival overgrowth in pediatric patients and its correlation with the dental plaque status.

BACKGROUND AND OBJECTIVES: Nucleolar organizer Regions (NORs) are situated within the nucleolus of a cell. The proteins are selectively stained by the silver colloid technique that is known as the AgNOR technique. AgNOR stain can be visualized as a black dot under the optical microscope. The present study aimed to evaluate the cases for quantitative estimation of AgNORs in the epithelial cells in various grades of gingival overgrowth to that of normal gingival tissues. MATERIALS AND METHODS: Only preadolescent and adolescent groups aged up to 14 years were selected. Twenty normal and 31 disease cases of gingival overgrowth were selected. The tissue sections were stained by the hematoxylin and eosin (HandE) technique for the routine histological evaluation, while the AgNOR counts were performed through the improved one-step method of Ploton et al. RESULTS: HandE staining revealed five different types of gingival overgrowth. The plaque index (PI), gingival index (GI), and AgNOR count were not significantly (P> 0.05) higher than that of control cases in pyogenic granuloma, puberty gingivitis, and in drug-induced gingival overgrowth cases. In gingival fibromatosis cases, for comparison of different indices t-tests were done. The PI when compared with AgNOR count was found significant at 5% level and 0.1% level for mixed and permanent dentition, respectively. The GI when compared with AgNOR count was found significant at 1% level and 0.1% level in mixed and permanent dentitions, respectively.

[Association of expression of progesterone and estrogen-alpha receptors with the activity of argyrophilic proteins in nucleolar organizer regions in smooth muscle tumors of the corpus uteri].

The immunohistochemical technique was used to study the levels progesterone and estrogen-alpha receptors in the smooth muscle cells, by examining the specimens taken from 168 leiomyosarcomas (LMS) and 106 leiomyomas (LM) of the corpus uteri. The presence and degree of receptor expression were shown to be related to the proliferative activity (PA) (Ki67 protein expression), the activity of argyrophilic proteins in the nucleolar organizer region (Ag-NOR-protein) (silver-plating with AgNO3, as described by Y. Daskal) and the proteins regulating the apoptotic system (Bcl-2 expression) (93.4 +/- 1.1%). The presence of sex hormone receptors in LMS was associated with low PA (8.5 +/- 3.3%), the decreased synthesis of Ag-NOR proteins (15.1 +/- 2.7%), and high Bcl-2 activity (93.4 +/- 1.1%) although the presence of the receptors had no significant effect on p53. The negative expression of both types of receptors was characteristic for LMS and associated with high PA (19.3 +/- 3.1%), the high expression of Ag-NOR-proteins (25.3 +/- 2.6%), and a drastic reduction in the amplification of Bcl-2 (38.4 +/- 10%) in reference to the positive receptor status. Receptor coexpression was associated with relatively low PA, the expression of argyrophilic proteins and the relatively high expression Bcl-2 without any noticeable impact on the expression of p53.

Demonstration of decreased activity of dorsal raphe nucleus neurons in depressed suicidal patients by the AgNOR staining method.

BACKGROUND: Suicide and depression are closely related yet distinct phenomena. In both these phenomena, research has focused on central serotonergic system disturbances. The dorsal raphe nucleus (DRN) is the main source of serotonergic innervation of limbic structures crucial for the regulation of emotionally influenced behaviour. METHODS: The study was carried out on paraffin-embedded brains from 23 depressed patients (12 suicides and 11 non-suicides) and 26 matched controls without mental disorders. The karyometric parameters of DRN neurons were evaluated by the AgNOR silver staining method. RESULTS: The significant effect of suicide on the nuclear area was found in the cumulative analysis of all DRN subnuclei (ANOVA, P=0.032). A decreased mean value of this parameter was observed in the suicides group versus controls (t-test, P=0.032). This effect was especially pronounced in the violent suicide victims (t-test, P=0.001), who also demonstrated a decreased AgNOR area versus controls (t-test, P=0.007). No significant effect of depression or polarity on AgNOR parameters was found. LIMITATIONS: A major limitation of this study is relatively small case number. A further limitation is given by the lack of data on drug exposure across the whole life span. CONCLUSION: Our findings suggest that hypoactivity of DRN neurons is a distinct phenomenon in depression, specific only for suicidal subgroup of depressed patients.

AgNOR cut-point to distinguish mild and moderate epithelial dysplasia.

BACKGROUND: Distinguishing mild and moderate epithelial dysplasia is difficult and controversial. We demonstrated earlier that silver-stainable nucleolar organizer region (AgNOR) is a simple, inexpensive and quantifiable method which can standardize this distinction reliably. OBJECTIVE: To establish a cut-point mean AgNOR count for epithelial dysplasia validated by bootstrapping. METHODS: One hundred oral leukoplakia biopsies at Dr R. Ahmed Dental College, Kolkata, India, examined for epithelial dysplasia using standard method (gold standard) were assessed for their mean AgNOR counts. A cut-point was selected employing receiver operating characteristic (ROC) curve. This estimate was stabilized by 10 000 resamples (with replacement) using parametric and non-parametric bootstrap, a Monte Carlo statistical method, corrected for bias to estimate standard errors (SE) of cut-point. RESULTS: Area under the curve (AUC) was 74%; non-parametric method suggested mean AgNOR cut-point = 2.42 AgNORs/nucleus; parametric method suggested cut-point = 2.57. The cut-point 95% confidence intervals (CIs) (bias corrected and accelerated) from parametric method (2.31; 2.66) was lower than non-parametric (2.43; 2.82). CONCLUSION: Applying a conservative paradigm, taking the lowest of all bootstrap 95% CIs, we suggest that mean AgNOR count of 2.3 be used as a cut-point for distinguishing between mild and moderate dysplasia. This strategy will provide an inexpensive, meaningful, definitive, reproducible and consistent diagnostic test for epithelial dysplasia.

Oxidant-induced decrease of the expression of nucleolar organizer regions in pig lymphocytes can be useful for monitoring the cellular effects of oxidative stress.

The technique of silver staining of nucleolar organizer regions (AgNORs) was chosen to estimate the transcriptionally active metaphase and interphase nucleolar organizer regions (NORs) in pig peripheral blood lymphocytes exposed to oxidative agents in vitro. The quantitative analysis of AgNORs was performed by using the counting method and the morphometric method. We found that hydrogen peroxide and 2,2'-azobis-(2-amidinopropane)-dihydrochloride (AAPH) induced a concentration-dependent decrease in NORs activity - in the case of metaphase the NORs activity was exclusively seen on chromosome pairs 8 - which can be considered as another estimate of cellular oxidative stress. Moreover, biomarkers of cyto- and genotoxicity, such as the percentage of apoptotic and necrotic cells, the nuclear division index (NDI) and formation of micronuclei (MN) were used to measure harmful effects provoked by the agents tested.

The diagnostic and prognostic implications of silver-binding nucleolar organizer regions in periodontal lesions.

BACKGROUND: The periodontal lesions with cellular proliferation can be assessed by various methods. One of the most recent methods to determine the proliferative activity is silver-staining nucleolar organizer region (AgNOR) staining. The purpose of the present study was to evaluate, if AgNOR count can act as a proliferative marker and can aid in the diagnosis and prognosis of periodontal lesions. MATERIALS AND METHODS: For this study, subjects with healthy gingival status, non-neoplastic lesions, neoplastic lesions, and plaque-induced gingivitis were included. Following the provisional diagnosis of clinical entity, biopsies were taken from the respective selected sites for histopathological diagnosis. In plaque-induced gingivitis cases, a second biopsy was taken from the selected sites 3 weeks following scaling. After histological confirmation, one more section was prepared, which was subjected to AgNOR staining, and AgNOR numbers were counted by individual and cluster counts and statistically analyzed. RESULTS: Results showed the highest AgNOR count in neoplastic lesions. Non-neoplastic lesions showed a higher AgNOR count as compared to clinically healthy gingiva. Plaque-induced gingivitis showed a considerable reduction in AgNOR count after treatment. CONCLUSION: Results of this study confirmed that AgNOR count reflects the cellular proliferation and has a limited diagnostic value. However, the prognostic value of AgNOR for periodontal lesions is dependable.