[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.

Complement C4 maintains peripheral B-cell tolerance in a myeloid cell dependent manner.

The factors that allow self-reactive B cells to escape negative selection and become activated remain poorly defined. Using a BCR knock-in mouse strain, we identify a pathway by which B-cell selection to nucleolar self-antigens is complement dependent. Deficiency in complement component C4 led to a breakdown in the elimination of autoreactive B-cell clones at the transitional stage, characterized by a relative increase in their response to a range of stimuli, entrance into follicles, and a greater propensity to form self-reactive GCs. Using mixed BM chimeras, we found that the myeloid compartment was sufficient to restore negative selection in the autoreactive mice. A model is proposed in which in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells, allowing the maturation and activation of self-reactive B-cell clones leading to increased spontaneous formation of GCs.

Human autoantibody to RNA polymerase I transcription factor hUBF. Molecular identity of nucleolus organizer region autoantigen NOR-90 and ribosomal RNA transcription upstream binding factor.

In dividing eukaryotic cells, nucleoli disperse before mitosis and reform in daughter cells at sites of ribosomal RNA (rRNA) gene clusters that are at the secondary constrictions of chromosomes, called nucleolus organizer regions (NORs). In this study, cDNA clones for a NOR autoantigen (NOR-90) were selected using a specific human autoantibody probe and were subsequently identified to encode an alternative form of the reported human upstream binding factor (hUBF). Results from immunoprecipitation showed that anti-NOR-90 antibodies recognized both forms of hUBF/NOR-90. Our data therefore showed that UBF, a critical factor in the regulation of rRNA transcription, was tightly bound to NOR during mitosis even when rRNA synthesis was thought to be minimal. Furthermore, we identified a nucleolar transcription factor as a novel target for human autoimmune response.

Leukocyte Nucleolus and Anisakis pegreffii-When Falling Apart Means Falling in Place.

The view of the nucleolus as a mere ribosomal factory has been recently expanded, highlighting its essential role in immune and stress-related signalling and orchestrating. It has been shown that the nucleolus structure, formed around nucleolus organiser regions (NORs) and attributed Cajal bodies, is prone to disassembly and reassembly correlated to various physiological and pathological stimuli. To evaluate the effect of parasite stimulus on the structure of the leukocyte nucleolus, we exposed rat peripheral blood mononuclear cells (PBMC) to the crude extract of the nematode A. pegreffii (Anisakidae), and compared the observed changes to the effect of control (RPMI-1640 media), immunosuppressive (MPA) and immunostimulant treatment (bacterial lipopolysaccharide (LPS) and viral analogue polyinosinic:polycytidylic acid (poly I:C)) by confocal microscopy. Poly I:C triggered the most accentuated changes such as nucleolar fragmentation and structural unravelling, LPS induced nucleolus thickening reminiscent of cell activation, while MPA induced disassembly of dense fibrillar and granular components. A. pegreffii crude extract triggered nucleolar segregation, expectedly more enhanced in treatment with a higher dose. This is the first evidence that leukocyte nucleoli already undergo structural changes 12 h post-parasitic stimuli, although these are likely to subside after successful cell activation.

NOR expression increases on metaphase chromosomes of Down syndrome lymphocytes in concordance with mitogen concentration in culture medium.

BACKGROUND: Regulation of nucleolus organizer region (NOR) expression in trisomy 21 (Down syndrome [DS]) cells is not fully explained. This work compared NOR expression on metaphase chromosomes in gradiently stimulated lymphocytes from DS patients with those from healthy controls. METHOD: Conventional peripheral blood culture (72 h) and chromosomal preparation procedures were used except that blood samples from each individual were cultivated in the same but gradiently increasing concentrations (0.37, 0.75, 1.48, and 2.21 ml) of phytohemagglutinin (PHA) per 100 ml of medium. One hundred consecutive metaphases per concentration were analyzed for scoring the means of the active NORs bearing chromosomes (AgNOR+ chromosome) per individual and per concentration. RESULTS: In contrast to healthy controls (n=24), AgNOR+ chromosomal number in lymphocytes from 30 DS patients increased in concordance to the gradient of PHA concentration in the culture medium. CONCLUSION: DS lymphocytes do not downregulate their NOR expression in the limit of control cells. This in vitro result may serve as a clue for the explanation of the DS phenotype due to the wasted energy in producing unnecessary rRNA transcripts and AgNOR proteins in utero during organogenesis. These results also indicate that precautions must be used in routine work of NOR evaluation/interpretation in DS lymphocytes.

Screening for autoantibodies to the nucleolar U3- and Th(7-2) ribonucleoproteins in patients' sera using antisense riboprobes.

In this study we report the detection of autoantibodies to the nucleolar U3- and Th(7-2) ribonucleoprotein (RNP) particles in sera from patients with connective tissue diseases. The method described employs radioactively labelled antisense U3- and Th RNA which are hybridized to immunoprecipitated U3- or Th RNA from a HeLa cell extract. Of the 66 sera that were screened with this method seven sera (11%) precipitated only Th RNP, 16 sera (24%) precipitated only U3 RNP and 4 sera (6%) precipitated both U3- and Th RNP. Both anti-U3 RNP and anti-Th RNP activity appeared to be mostly associated with scleroderma or scleroderma-associated diseases. Using this method we also showed that some of the Th RNP particles in a cell extract are associated with the La autoantigen. We conclude that for the identification of immunoprecipitated RNAs this method is very sensitive and provides unambiguous data.

The two isoforms of the 90-kDalton nucleolus organizer region autoantigen (upstream binding factor) bind with different avidity to DNA modified by the antitumor drug cisplatin.

It has been previously described that some proteins containing HMG boxes are able to bind more strongly to DNA modified with cis-diamminedichloroplatinum (II) (cisplatin) than to unmodified DNA. In the present study, we analyzed the interaction of cisplatin-modified DNA with the human autoantigen NOR-90 (UBF), a transcription factor that contains several HMG boxes. Using autoantibodies against NOR-90 to perform ELISA and immunoprecipitation, it was confirmed that NOR-90 (UBF) was able to bind cisplatin-modified DNA more avidly than unmodified DNA or trans-diamminedichloroplatinum(II) (transplatin) modified DNA. Moreover, by Southwestern, we observed that the 97 kDalton isoform of NOR-90 (UBF1) was able to bind cisplatin-modified DNA more strongly than the 94 kDalton isoform (UBF2); binding of unmodified DNA or transplatin-modified DNA was not detected with either isoform. Sera containing autoantibodies against NOR-90 did not inhibit, but increased the binding of NOR-90 to cisplatin-modified DNA.

Breast carcinoma kinetics. Argyrophilic nucleolar organizer region counts correlate with Ki67 scores.

This study assessed the value of argyrophilic nucleolar organizer region (AgNOR) staining as a potential technique for the estimation of cell kinetics in conventional histology sections, in benign and malignant breast lesions. Using a silver staining technique and immunohistochemistry, the authors correlated the numbers of argyrophilic nucleolar organizer regions (AgNORs) and Ki67 scores in 70 breast carcinomas and 27 benign breast lesions. Epithelial cells in fibrocystic disease and fibroadenomas contained a mean of 2.65-6.8 small uniform AgNORs per cell, whereas malignant cells contained 4.6-26.9 frequently highly irregular AgNORs. In benign tissue, Ki67 scores ranged from 0 to 4%; in malignant tumors, Ki67 scores ranged from 3.0 to 98%. The correlation between AgNOR counts and Ki67 scores was highly significant (P less than 0.001). The authors concluded that AgNOR counts performed on routine formalin-fixed paraffin sections furnish significant kinetic information. Furthermore, the difference in AgNOR counts between benign and malignant tumors is such that they may be of diagnostic value.

Chromosome banding analysis of a new stemline of hypodiploid Hirosaki sarcoma established by single cell transplantation.

A new hypodiploid stemline of the long-lasting ascites sarcoma, Hirosaki sarcoma (HS), was established by means of single-cell transplantation, and its G-, C- and NOR-banding patterns were compared with those of the predecessor stemline, with special notice taken of the structural alteration of marker chromosomes. Their chromosomal relationship was well explained by the rearrangement, t(M7pM8q): Translocation of the short arm of marker M7 to the NOR of marker M8 produced a new marker M8t, and the translocation-carrying variant cells grew into a subline. A new translocation-carrying stemline was formed from this subline by single-cell transplantation. Judging from the size and number of silver-positive NORs, the NOR activity was significantly higher in this stemline than in its predecessor. The increased NOR activity might have been partly responsible for the growth of a translocation-carrying variant cell into a new subline. Single-cell-mass transplantation may be one of the most effective systems for studying the proliferative ability of subline cells and for understanding the mechanism of stemline formation in transplantable rat ascites sarcomas.

Prognostic implications of proliferating cell nuclear antigen (PCNA), AgNORs and P53 in non-Hodgkin's lymphomas.

We investigated the prognostic value of proliferating cell nuclear antigen (PCNA) and p53 oncoprotein expression and of nucleolar organiser region (NOR) scoring, in relation to classic clinicopathological parameters, in a series of non-Hodgkin's lymphomas (NHL). Paraffin embedded tissue from 91 patients with NHL was stained immunohistochemically with the monoclonal antibodies PC-10 (PCNA) and DO-1 (p53) and histochemically with the AgNOR technique. The median follow-up was 48 (4 to 193) months. The impact of PCNA and p53 expression and of AgNOR number on survival was tested using univariate as well as multivariate analysis, in order to circumvent the heterogeneity in histologic grade, type and therapy. Univariate analysis identified seven variables related to overall survival: histologic type and grade, clinical stage, chemotherapy, p53 labelling index (LI), PCNA LI and AgNOR score, whereas only one parameter i.e. histologic grade influenced disease-free survival. In multivariate analysis stage, PCNA LI and AgNOR score predicted independently overall survival. PCNA was also the only independent predictor of post-relapse survival and histologic grade the most important indicator of disease-free survival. In conclusion, PCNA expression and AgNOR number may be better predictors of overall and post-relapse survival than histologic grade. The latter remains the most valuable prognostic indicator of disease-free survival.

Differential diagnosis of fine needle aspiration smears of thyroid nodules. Cytologic features and AgNORs.

OBJECTIVE: To determine the value of cytomorphologic features and argyrophilic nucleolar organizer regions in fine needle aspiration smears of thyroid nodules. STUDY DESIGN: Cytomorphologic features were analyzed for frequency and predictive value in smears of 60 histologically confirmed cases of thyroid nodules, comprising 16 cases of papillary carcinoma, 19 of follicular adenoma, 5 of follicular carcinoma, 3 or medullary carcinoma and 17 of adenomatous goiter. The smears were also stained for AgNORs to compare AgNOR counts in these lesions. RESULTS: Intact follicles and abundant background colloid were useful for the diagnosis of adenomatous goiter; metaplastic cells, nuclear grooves and multinucleate giant cells predicted the diagnosis of papillary carcinoma; and acinar formations were important in the diagnosis of follicular adenoma and follicular carcinoma. AgNOR counts showed major overlaps among the various groups, although the mean counts in neoplastic lesions were slightly higher than those in adenomatous goiter CONCLUSION: AgNOR counts failed to have any significant diagnostic utility, and the fine needle aspiration cytology diagnosis of thyroid nodules must still be based on a combination of cytologic features.

Feline lymphoma (145 cases): proliferation indices, cluster of differentiation 3 immunoreactivity, and their association with prognosis in 90 cats.

Paraffin-embedded, formalin-fixed tissue samples from 145 cats with lymphoma were analyzed for cluster of differentiation 3 (CD3, a surface antigen) immunoreactivity, argyrophilic nucleolar organizer region (AgNOR) frequency, and proliferating cell nuclear antigen labeling index (PCNA-LI). This information along with signalment, anatomic site, and feline leukemia virus (FeLV) antigen status was used to determine the potential of these indicators to predict response to therapy, remission, and survival times, and to characterize cats with lymphoma in the era of general availability of FeLV testing and vaccination. Alimentary lymphoma, primarily occurring in older, FeLV-negative cats, was the most common site of involvement. Although the majority of tumors from FeLV-positive cats were CD3-immunoreactive, only one half of CD3-immunoreactive tumors occurred in FeLV-positive cats. Median remission duration and survival times were 126 days and 143 days, respectively, for all cats. Measures of tumor cell proliferation (AgNOR frequency and PCNA-LI) and CD3-immunoreactivity were not predictive of outcome. When all prognostic factors were accounted for by multivariate analysis, response to therapy, FeLV status, and clinical substage were predictive of outcome. FeLV-negative cats that achieved a complete response following induction therapy were likely to have durable (i.e., > 6-month) responses, particularly when doxorubicin was included in the chemotherapy protocol. However, FeLV-positive cats had significantly shorter remission and survival times with available chemotherapeutic protocols.

Lewis antigens and argyrophilic nucleolar organizer regions staining for assessment of potential malignancy of adenomatous polyps of the gastrointestinal tract in children.

Adenomatous polyps (AP) of the gastrointestinal tract in children are very rare. Because of their potential malignancy, they are of great clinical importance. There is little experience in the management of children with AP. The immunohistochemical expression of the Lewis blood group antigens (BGA) (sialosyl-Le(a), Le(a), Leb, Le(x), and Le(y)) and the number of activated nucleoli with the silver staining method for nucleolar organizer regions (AgNORs) were studied in two children with AP. In a girl with isolated AP of the stomach and colon, it was found that antigens Le(b) and s-Le(a) were expressed extensively in the gastric adenoma, and sialosyl-Le(a) throughout the entire length of the rectal adenoma crypts, but in the AgNORs stain the number of nucleoli ranged from two to four, evidencing changes of a benign character. In the case of familial adenomatous polyposis diagnosed in a 9-year-old boy, in some colonic adenomas the number of activated nucleoli was greater than five, and the Le(b) antigen was expressed in superficial epithelial cells in one of the adenomas. Also, extensive expression of antigens Le(y) and s-Le(a) throughout the entire length of the crypt in another polyp removed was observed. We believe that immunohistochemical study of the intensity and extent of the expression of Lewis BGA in the polyp tissue simultaneously with the determination of the number of activated nucleoli by the AgNORs staining method can be helpful in better analysis of cytological risk factors of a malignant transformation.

The prognostic value of proliferating cell nuclear antigen, Ki-67 and nucleolar organizer region in transitional cell carcinoma of the bladder.

OBJECTIVES: To investigate the value of proliferating cell nuclear antigen (PCNA), Ki-67 antigen labelling indices and nucleolar organizer region (NOR) score in relation to histological grade, stage, recurrence and progression of the bladder tumor. MATERIALS AND METHODS: Tissue specimens from 77 bladder cancer patients (43 superficial, 34 invasive) were immunostained with PCNA and Ki-67 and stained with AgNOR. Thirteen specimens of normal bladder mucosa served as controls. RESULTS: In comparison to normal bladder mucosa the values of the three indicators were significantly greater (p < 0.001). There was a significant relationship between PCNA, Ki-67 indices, AgNOR scores and grade and stage of the tumor (p < 0.001). All indicators also correlated with each other (p < 0.001). The Kaplan-Meier curves for recurrence-progression free survival revealed that patients with a PCNA labelling index >36.22%, Ki-67 labelling index >29.68% and AgNOR score > 3.34 had a worse prognosis than those with <36.22%, <29.68% and <3.34, respectively. CONCLUSIONS: PCNA, Ki-67 indices and AgNOR scores correlated with each other and with tumor grade and stage. These proliferation markers may give objective and accurate information about the biological behavior of bladder transitional cell carcinoma.

[Research on the regular pattern of Ag-NOR changes in patients with cancer].

OBJECTIVE: To correlate the relationship between rDNA transcriptional activity of CD4+ helper T cells and immunity in patients with cancer through silver stainability of nucleolus organizing regions (Ag-NORs). METHODS: The Th(CD4+) rDNA transcriptional activity was compared among patients with cancer, infectious diseases, non-cancerous non-infectious diseases through image analysis. RESULTS: Th(CD4+) rDNA transcriptional activity was decreased extremely in patients with cancer, but increased in patients with inflammation compared with normal controls. CONCLUSION: The analysis of Th(CD4+) rDNA transcriptional activity may serve as a new tumor marker for tumor diagnosis and differential diagnosis. Therefore, it may provide an innovative and reliable method for clinical cancer diagnosis.

[Frequency of acrocentric chromosomal associations and their silver staining in human lymphocytes in immune reactions]. / Chastota assotsiatsii akrotsentricheskikh khromosom i ikh okrashivanie serebrom v limfotsitakh krovi cheloveka pri immunnykh reaktsiiakh.

Ag-staining of the nucleolar organizer regions of acrocentric chromosomes of T-lymphocytes did not change during the immune response in children with porotitis and in those being in contact with parotitis-suffering children, as well as in young adults previously immunized by staphylococcal anatoxin. This character displayed individual peculiarities. No differences in these age groups were detected. A positive correlation was found between the size of Ag-band and the ability of chromosomes to make associations. Ag-staining and participation of G-chromosomes in associations was higher than those markers in D-chromosomes.

[The immunolocalization of the ribosomal gene transcription initiation factor UBF in the interphase and mitosis]. / Immunolokalizatsiia faktora initsiatsii transkriptsii ribosomnykh genov UBF v interfaze i mitoze.

Serum P419 from a patient with rheumatoid arthritis with a high specificity immunolabeling nucleoli in various mammalian cells has been identified. On the Western blots of total cellular proteins or proteins extracted from isolated nucleoli it cross-reacted with a doublet of polypeptides of 97 and 94 kDa. That is why this serum has been concluded to recognize UBF, or RNA polymerase I-specific transcription initiation factor. It was shown that UBF remained bound to the nucleoli or nucleolus organizing regions (NORs) of mitotic chromosomes despite the level of rDNA transcription. Nevertheless, intranucleolar localization of UBF was dramatically changed after partial or complete block of rRNA synthesis. In pycnotic cells positive labeling was found within the whole nucleus and cytoplasm instead of nucleolus. In metaphase UBF molecules are unequally distributed between the particular NORs, whereas in anaphase they are uniformly allocated between the daughter cells.

Anti-NOR 90. A new autoantibody in scleroderma that recognizes a 90-kDa component of the nucleolus-organizing region of chromatin.

We identified a patient (CAG) with scleroderma whose serum contained a high titer of IgG class antibodies that stained nucleoli in a pattern of independent tiny spots. When tested on isolated chromosomes, these antibodies selectively stained the nucleolus-organizing regions (NOR) of chromosomes 13, 14, 15, 21, and 22. These staining patterns were not altered when substrate cells and chromosomes were treated with RNase, 0.1 M HC1, or 4 M urea, but they were abolished by treatment with DNase and trypsin. Immunoblots performed with serum CAG on isolated nucleolar substrates identified a protein antigen of approximately 90 kDa. Antibodies affinity-purified from this protein selectively stained nucleoli and NOR chromosomal regions. Therefore, this protein is the antigen that accounts for the ability of serum CAG to recognize the NOR. In a search for the NOR 90-kDa specificity among 254 patients with various rheumatic diseases, we found nine additional patients whose sera stained metaphase chromosomes selectively at the NOR. Sera from five of them (three with scleroderma, two of unknown diagnosis) recognized a protein that electrophoretically co-migrated with the CAG antigen. Thus, scleroderma is present in at least four of six who appear to have this specificity. We conclude that autoantibodies to the NOR 90-kDa antigen have an association with scleroderma and may be useful diagnostically and as a probe for further studies of the biology of the cell nucleolus.

Correlation of morphological patterns of nucleoli in alveolar macrophages with HLA-DR antigen expression in sarcoidosis.

BACKGROUND: Alveolar macrophages from patients with sarcoidosis express increased quantities of HLA-DR during activation. Because silver staining has been described as a sensitive indicator of cellular activity a study was performed to examine whether it relates to HLA-DR antigen expression. METHODS: The relation between silver staining patterns of nucleoli and HLA-DR antigen expression was examined in alveolar macrophages collected by bronchoalveolar lavage from 11 patients with pulmonary sarcoidosis and 11 control subjects. RESULTS: The mean (SD) number of silver stained protein dots associated with the nucleolar organiser regions (AgNORs) was significantly higher in alveolar macrophages from patients with sarcoidosis (7.5 (1.5)) than in those from control subjects (5.6 (0.6)). The number of silver stained dots in alveolar macrophages correlated significantly with the intensity and the density of HLA-DR antigen expression in the patients with sarcoidosis. CONCLUSIONS: Silver staining may be a sensitive tool for the investigation of the biological cell activity of alveolar macrophages in sarcoidosis.