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.

[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.

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.

The association of antinuclear antibodies with organ involvement and survival in systemic sclerosis.

OBJECTIVES: To study the frequency and specificity of antinuclear antibodies (ANA) and their association with internal organ involvement and survival in systemic sclerosis (SSc). METHODS: Sera from 276 SSc patients were analysed by an indirect immunofluorescence (IIF) technique with HEp-2 cells as a substrate to categorize centromeric (ACA), nucleolar, speckled and homogeneous nuclear IIF patterns. Specific ANA were determined as follows: anti-DNA topoisomerase I (anti-topo I) by double immunodiffusion, anti-U1 RNP by passive haemagglutination, anti-RNA polymerase I, II and III (anti-RNAP) and anti-histone (AHA) antibodies by enzyme immunoassays. During the follow-up of 7.0+/-4.5 (mean+/-S.D.) yr the occurrence of clinical manifestations and internal organ involvement was registered. RESULTS: ANA were present in 84% of the patients. The most common patterns of the IIF were speckled (41%), homogeneous (25%), nucleolar (24%) and centromeric (18%). A nucleolar pattern was associated with pulmonary fibrosis (P < 0.01) and cardiomegaly (P < 0.05). ACA were related to organic vasculopathy (P < 0.05) and renal involvement (P < 0.01), but not to pulmonary fibrosis (P < 0.01). Anti-topo I were present in 9.4%, anti-U1 RNP in 21%, anti-RNAP in 22% and AHA in 16% of the patients. Pulmonary involvement was more common in patients with anti-topo I (P < 0.05), whereas AHA-positive patients were characterized by cardiac (P < 0.05), pulmonary (P < 0.05) and renal (P < 0.05) involvement. A nucleolar IIF pattern and AHA were both associated with a decreased survival [relative risk of death 1.71 (P < 0.05) and 2.36 (P < 0.01), respectively]. CONCLUSIONS: AHA and a nucleolar HEp-2 cell pattern may indicate critical organ involvement and predict a reduced survival in SSc patients.

Expression of topoisomerase IIalpha, Ki-67, proliferating cell nuclear antigen, p53, and argyrophilic nucleolar organizer regions in vulvar squamous lesions.

OBJECTIVE: It was the aim of this study to investigate the expression of topoisomerase IIalpha (topo IIalpha), Ki-67, proliferating cell nuclear antigen (PCNA), p53, and argyrophilic nucleolar organizer region (AgNOR) staining in normal vulvar epithelia (NE, N = 10), vulvar condylomas (VC, N = 24), vulvar intraepithelial neoplasia (VIN, N = 26), as well as squamous cell carcinomas (SCC, N = 22) of the vulva. METHODS: Formalin-fixed, paraffin-embedded archival tissue sections were immunostained with monoclonal antibodies against topo IIalpha, p53, and PCNA, as well as an affinity-isolated prediluted ready-to-use Ki-67 antibody using a standard immunohistochemical method, and stained with a colloid silver solution for AgNORs. Immunostaining was quantitated by determining the percentage of positively staining nuclei in each sample to express the labeling indices (LIs) by counting the immunoreactive nuclei in 1000 epithelial cells per case for each antibody. In each specimen 200 nuclei were examined using a x100 oil emersion lens, and the mean number of AgNORs per nucleus (AC) was calculated. RESULTS: The LIs for topo IIalpha, Ki-67, and PCNA as well as ACs increased stepwise from NE to VCs, VIN lesions, and SCCs. In contrast to PCNA LIs and ACs, a consistent correlation in all four groups was found for Ki-67 and topo IIalpha, suggesting that the latter is a proliferation-associated marker in these tissues. p53 expression was seen 8.3% of VCs, 30.8% of VIN lesions, and 54.45% of SCCs. p53 LIs were not correlated with LIs for topo IIalpha or Ki-67 in SCCs. The LIs for topo IIalpha, Ki-67, PCNA, p53, and ACs were not related to tumor progression, FIGO stage, or tumor grade in SCCs. CONCLUSIONS: This study presents topo IIalpha and Ki-67 as useful proliferation-associated markers of vulvar epithelia.

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.

Autoantibodies to NOR 90/hUBF: longterm clinical and serological followup in a patient with limited systemic sclerosis suggests an antigen driven immune response.

We describe the clinical and serological followup of a 9-year-old girl with anti-nucleolar organizing region 90/human upstream-binding factor (anti-NOR 90/hUBF) who had features of systemic sclerosis over a period of 17 years, from childhood into adulthood. We review the associations of anti-UBF autoantibodies, and provide evidence that anti-NOR 90/UBF immune response is antigen driven.

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.

Dynamic of nucleolus organizer regions and karyotype evolution in Indian pygmy field mice.

Ag-NOR staining and fluorescence in situ hybridization with rDNA probes showed an unusually high number of NORs in the Indian pygmy field mice, Mus booduga and the M. terricolor complex. The chromosomal location of the NORs was also altered in terricolor, they were shifted from the proximal regions of the long arms to the tips of the perceptible heterochromatic short arms of the acrocentric autosomes. The results suggested dispersion of the NORs in the booduga-terricolor lineage probably by transposition, and relocalization of the NORs in the terricolor complex by centric reorganization during the process of replacement of the Mus musculus-related AT-rich heterochromatin with the terricolor-specific heterochromatin.

[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.

Study of borderline and invasive mucinous ovarian tumors using Ki-67 (MIB 1) antibodies and nucleolar organizer region (NOR) staining.

Extensive application of new methods in histopathology has resulted in large quantities of data on molecular markers in different types of human tumors. The main purpose has been to obtain additional parameters for the characterization of various types of malignancy to give more precise information on their biological behavior. In this study we tested AgNOR and Ki-67 (MIB 1) staining in 39 ovarian mucinous tumors to evaluate its diagnostic potential in distinguishing between borderline tumors and well-differentiated carcinomas at stage I of FIGO classification. In general, there was an increase in AgNOR median values from benign (1.83 +/- 0.8) to well-differentiated carcinomas (2.91 +/- 1.3) with an intermediate value in borderline (2.3 +/- 1.6). The lowest Ki-67 labeling index (LI) was found in benign cystadenomas (5.7% +/- 3.4%) and papillary mucinous cystadenomas (7.9 +/- 5.8%). In borderline tumors mean LI was 12.2 +/- 10.9% whereas well-differentiated carcinomas had higher LI (16.8 +/- 10.2%). There were no statistically significant differences between the evaluated tumors and techniques applied. In our opinion the AgNOR count and Ki-67 LI do not appear useful for assessing differences between borderline and well-differentiated mucinous ovarian tumors at stage I of clinical advancement.

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.

Autoantibodies to components of the mitotic apparatus.

Autoantibodies directed to a variety of cellular antigens and organelles are a feature of autoimmune diseases. They have proven useful in a clinical setting to establish diagnosis, estimate prognosis, follow disease progression, alter therapy, and initiate new investigations. Cellular and molecular biologists have used autoantibodies as probes to identify molecules involved in key cellular processes. One of the most interesting sets of autoantibodies are those that target antigens within the mitotic apparatus (MA). The MA includes chromosomes, spindle microtubules and centrosomes. The identification, localization, function, and clinical relevance of MA autoantigens is the focus of this review.

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.

Detection of autoantibodies to nucleolar transcription factor NOR 90/hUBF in sera of patients with rheumatic diseases, by recombinant autoantigen-based assays.

OBJECTIVE: We attempted to clarify the clinical characteristics of Japanese patients with autoantibodies to nucleolar transcription factor NOR 90/hUBF (anti-NOR 90) and to analyze the autoantigenic epitopes recognized by anti-NOR 90. METHODS: Ninety-one patient sera containing anti-nucleolar antibodies (ANoA) by indirect immunofluorescence were collected. Immunoblottings were performed using recombinant fusion proteins expressed from several cloned complementary DNA (cDNA) encoding the NOR 90/hUBF autoantigen. RESULTS: Anti-NOR 90 were detected in sera from 9 (9.9%) of 91 patients with ANoA. Seven of these patients were diagnosed as having Sjögren's syndrome, 4 had concomitant rheumatoid arthritis, 1 had concomitant systemic sclerosis (SSc), and 2 had SSc alone. All 9 sera were reactive with more than 2 recombinant fusion proteins from cDNA encoding separate regions on the hUBF polypeptide. CONCLUSION: The results suggest that while anti-NOR 90 antibodies are rare, they are associated with Sjögren's syndrome in Japanese patients, and that autoimmunity is targeted toward at least 2 separate regions (amino acids 89-310 and 310-633) of the hUBF polypeptide.

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.