Nucleolar Staining Research Articles

Nucleolar casein kinase 2 alpha as a prognostic factor in patients with surgically resected early‑stage lung adenocarcinoma.

Lung cancer remains a leading cause of global cancer‑related deaths, therefore the identification of prognostic factors for lung cancer is critical. Casein kinase 2 alpha (CK2α) is one of the driver kinases in various cancers, and it was previously demonstrated that CK2α localization was associated with a poor prognosis in invasive breast cancer. In the present study, the importance of CK2α in the nucleolus was explored as a potential prognostic marker for surgically resected early‑stage lung adenocarcinoma. The present study included 118 patients who underwent pulmonary lobectomy between 2014 and 2018 in Fukushima Medical University Hospital (Fukushima, Japan), and in whom CK2α localization in tumor samples was assessed by immunohistochemistry. Patient and tumor characteristics, including pathological stage, histological type and histological grade, were analyzed. Recurrence‑free survival (RFS) and overall survival were evaluated in relation to nucleolar CK2α staining. CK2α staining in the nucleoli was observed in 50.8% of lung adenocarcinoma tumors. Positive nucleolar CK2α staining was independent of pathological stage, histological type and histological grade. Patients with positive nucleolar CK2α staining exhibited significantly worse RFS compared with patients with negative staining. Multivariate analysis identified nucleolar CK2α staining and lymph node metastasis as independent poor prognostic factors. The results of the present study suggested that nucleolar CK2α staining is a novel and independent prognostic factor in surgically resected early‑stage lung adenocarcinoma. These findings indicated the potential of nucleolar CK2α as a predictive biomarker for future recurrence, and a guide to treatment decisions. Further research is required, particularly in understanding the molecular mechanisms linking nucleolar CK2α to recurrence.

Thioflavin-T: application as a neuronal body and nucleolar stain and the blue light photo enhancement effect

Thioflavin-T (THT) is a common and indispensable tool for the study of amyloid pathologies and protein aggregation, both in vitro and for histological samples. In this study we expand the use of THT beyond its canonical usage for staining amyloid plaques and demonstrate its novel use as an easy and rapid stain comparable to fluorescent Nissl staining, allowing for clear discernment of neuronal cell bodies and also nucleoli in fixed tissue and live cells. We believe that this is of value for any lab that studies central nervous system (CNS) tissues. Furthermore, we show that THT could potentially be used as a an alternative to the use of fluorescent reporters or other more costly RNA binding compounds in the study of nucleolar dynamics owing to its ability to clearly stain nucleoli in live cells. We also discovered the previously unreported effect of blue light exposure on the photo enhancement of THT excited by a 488 nm laser in stained tissue sample and how to avoid complications arising from this effect. Finally, we provide a simple protocol that can be easily adjusted either for using THT as a neuronal cell body and nucleoli stain, compatible with antibody based staining methods tested up to 4 fluorophores, or alternatively by using an additional washing step the protocol may be used for amyloid plaque detection in fixed brain tissue.

Karyotype Variability in Wild Narcissus poeticus L. Populations from Different Environmental Conditions in the Dinaric Alps.

Narcissus poeticus L. (Amaryllidaceae), a facultative serpentinophyte, is a highly variable species and particularly important ancestor of cultivated daffodils, but is rarely studied in field populations. This study, based on natural populations in the Balkans, focused on karyotype variability, genome size, ploidy and the presence of B chromosomes. Thirteen native populations from different environmental and soil conditions were collected and analyzed using flow cytometry to estimate nuclear genome size, fluorescence in situ hybridization (FISH) for physical mapping of rDNA, fluorochrome labeling (chromomycin and Hoechst) for heterochromatin organization and silver nitrate staining of nucleoli for determining rRNA gene activity. The organization of rDNA and natural triploids is reported here for the first time. The presence of individuals with B chromosomes (in 9/13 populations) and chromosomal rearrangements was also detected. The observed B chromosome showed three different morphotypes. The most frequent submetacentric type showed four different patterns, mainly with active ribosomal genes. The results obtained show that N. poeticus has a dynamic genome with variable genome size due to the presence of polyploidy, B chromosomes and chromosomal rearrangements. It is hypothesized that the observed changes reflect the response of the genome to different environmental conditions, where individuals with B chromosomes appear to have certain adaptive advantages.

Nucleolus imaging based on naphthalimide derivatives

Nucleolus was an important cellular organelle. The abnormal morphology and number of the nucleolus have been considered as diagnostic biomarkers for some human diseases. However, the imaging agent based on nucleolus was limited. In this manuscript, a series of nucleolar fluorescent probes based on naphthalimide derivatives (NI-1 ∼ NI-5) had been designed and synthesized. NI-1 ∼ NI-5 could penetrate cell membranes and nuclear membranes, achieve clear nucleolar staining in living cells. These results suggested that the presence of amino groups on the side chains of naphthalimide backbone could enhance the targeting to the cell nucleolus. In addition, the molecular docking results showed that NI-1 ∼ NI-5 formed hydrogen bonds and hydrophobic interactions with RNA, and exhibited enhanced fluorescence upon binding with RNA. These results will provide favorable support for the diagnosis and treatment of nucleolus-related diseases in the future.

Rapid, Economic, Acetic acid, Papanicolaou (REAP) staining technique analogous to the routine Papanicolaou staining (PAP) technique

The current research compared classical Papanicolaou stain with REAP (Rapid, Economic, Acetic Acid Papanicolaou) stain to evaluate staining character, range, charge virtue, and sustaining of stain. The PAP Stain is the standard dye in support of shielding cell study for any cell study and is used in various research centers with several reductions. In certainty, PAP Stain has lowered squamous cell carcinoma incidence by 70%, particularly within wealthy provinces with effectively-designed broadcasting systems. But the stain has gone through several changes, moving from the uncreative formal mechanism -> gradual REAP PAP staining in which staining duration level is decreased, it improves the quality, it is cost-effective, easily available, and ethyl alcohol is temporarily superseded with 1% ethanoic acid in advanced nations with guarding programs with careful planning. It has been used to access FNAC (fine needle aspiration cytology). Because of its precise nuclear and nucleoli staining, PAP staining of FNAC and fluid smears is a trustworthy stain. FNAC is a straightforward, speedy, and reliable pre-operative assessment and diagnosis process that has sparked advancements in dyeing technology which utilizes minimal coloring count with a clear cell framework. Around 40 minutes are needed for the standard methodology for traditional PAP Stain. REAP Stain was developed to address this, improving quality while cutting staining time to about one minute. This study's objective was to evaluate the value of the Rapid-Pap stain utilizing quantifiable investigation. The effectiveness of cytoplasmic and nuclear staining, stain preservation, value, and overall course of action for the two techniques—conventional PAP stain and REAP stain—were collated. When compared to traditional Papanicolaou, REAP stain offers a good, quick, and affordable replacement for the screening of cells. The REAP technique also does a superb job for the maintenance of stains.

Development of Highly Fluorogenic Styrene Probes for Visualizing RNA in Live Cells.

Styrene dyes are useful imaging probes and fluorescent sensors due to their strong fluorogenic responses to environmental changes or binding macromolecules. Previously, indole-containing styrene dyes have been reported to selectively bind RNA in the nucleolus and cytoplasm. However, the application of these indole-based dyes in cell imaging is limited by their moderate fluorescence enhancement and quantum yields, as well as relatively high background associated with these green-emitting dyes. In this work, we have investigated the positional and electronic effects of the electron donor by generating regioisomeric and isosteric analogues of the indole ring. Select probes exhibited large Stokes shifts, enhanced molar extinction coefficients, and bathochromic shifts in their absorption and fluorescence wavelengths. In particular, the indolizine analogues displayed high membrane permeability, strong fluorogenic responses upon binding RNA, compatibility with fluorescence lifetime imaging microscopy (FLIM), low cytotoxicity, and excellent photostability. These indolizine dyes not only give rise to rapid, sensitive, and intense staining of nucleoli in live cells but can also resolve subnucleolar structures enabling highly detailed studies of nucleolar morphology. Furthermore, our dyes can partition into RNA coacervates and resolve the formation of multiphase complex coacervate droplets. These indolizine-containing styrene probes offer the highest fluorescence enhancement among the RNA-selective dyes reported in the literature; thus, these new dyes are excellent alternatives to the commercially available RNA dye, SYTO RNASelect, for visualizing RNA in live cells and in vitro.

Julolidine-based small molecular probes for fluorescence imaging of RNA in live cells.

Intracellular RNA imaging with organic small molecular probes has been an intense topic, although the number of such reported dyes, particularly dyes with high quantum yields and long wavelength excitation/emission, is quite limited. The present work reports the design and synthesis of three cationic julolidine-azolium conjugates (OX-JLD, BTZ-JLD and SEZ-JLD) as turn-on fluorescent probes with appreciably high quantum yields and brightness upon interaction with RNA. A structure-efficiency relationship has been established for their potential for the interaction and imaging of intracellular RNA. Given their chemical structure, the free rotation between the donor and the acceptor gets restricted when the probes bind with RNA resulting in strong fluorescence emission towards a higher wavelength upon photoexcitation. A detailed investigation revealed that the photophysical properties and the optical responses of two probes, viz. BTZ-JLD and SEZ-JLD, towards RNA are very promising and qualify them to be suitable candidates for biological studies, particularly for cellular imaging applications. The probes allow imaging of intracellular RNA with prominent staining of nucleoli in live cells under a range of physiological conditions. The results of the cellular digest test established the appreciable RNA selectivity of BTZ-JLD and SEZ-JLD inside the cellular environment. Moreover, a comparison between the relative intensity profile of SEZ-JLD before and after the RNA-digestion test inside the cellular environment indicated that the interference of cellular viscosity in fluorescence enhancement is insignificant, and hence, SEZ-JLD can be used as a cell membrane permeable cationic molecular probe for deep-red imaging of intracellular RNA with a good degree of selectivity.

P24 A case of limited cutaneous systemic sclerosis evolving into an overlap syndrome with systemic lupus erythematosus and pyoderma gangrenosum

P24 A case of limited cutaneous systemic sclerosis evolving into an overlap syndrome with systemic lupus erythematosus and pyoderma gangrenosum

CK2α as a prognostic factor in invasive ductal carcinomas of the breast: cancer recurrence prognosis by surgical sampling

Breast cancer is the most common cancer in Japanese and Asian women. Although the 5-year survival rate of these patients is relatively high, recurrences and fatalities do occur. The objective of this work was to develop a reliable prognostic indicator to guide therapeutic intervention without waiting for recurrence or metastasis. We have previously shown that positive immunohistochemical staining of a protein kinase, CK2α (CSNK2A1), in nucleoli of invasive ductal carcinoma cells is strongly associated with tumour recurrence and poor patient outcomes. For this study, we did an immunohistochemistry investigation of the location of CK2α in the nuclei and nucleoli of formalin-fixed, paraffin-embedded tissue block samples from patients with invasive ductal carcinoma who underwent surgery in 2008-14 but no neoadjuvant chemotherapy at Hoshi General Hospital, Fukushima, Japan. Furthermore, a new study with 59 patients with luminal type breast cancer was done. Patients were followed-up (clinicopathological information and outcomes) for more than 5 years. To characterise CK2α-associated proteins systematically, nuclear and nucleolar extracts were prepared from MCF-7 cells and immunoprecipitated with anti-CK2α antibodies followed by nano-flow liquid chromatography mass spectrometry analysis. We did a Kaplan-Meier analysis to evaluate whether nucleolar CK2 staining might be associated with poor recurrence-free survival. This retrospective study was approved by the Institutional Review Boards of Fukushima Medical University and Hoshi General Hospital. Samples from 112 patients were included. 105 (94%) of 112 patients with invasive ductal carcinoma had high CK2α expression in nuclei, and 41 (37%) of 112 displayed it predominantly in nucleoli. Clinical and pathological malignancy was strongly correlated with high nuclear and nucleolar CK2α expression. Recurrence-free survival was significantly reduced in patients with positive nucleolar CK2α staining (n=41; median 7·89 years [IQR 4·86-8·86]), comparing with patients with negative nucleolar staining (n=71; median 8·89 years [7·87-9·79]; p<0·0001). Kaplan-Meier analysis showed a decrease of approximately 50% in recurrence-free survival in patients with nucleolar CK2α-positive stain in the triple-negative breast cancer (p=0·0069) and post-operative stage 3 (p=0·0073) groups. By contrast, no patients who relapsed or died in the triple-negative breast cancer group had negative staining for nucleolar CK2α. Evaluation of nucleolar CK2α can be considered as an independent prognostic factor because it reached significance in multivariate analysis (hazard ratio [HR] 5·26 [95% CI 1·35-20·55]; p=0·017). In addition, the HR of nucleolar CK2α-positive staining was less affected by adjustments in Cox proportional multivariate analysis. Cluster analysis showed that nucleolar CK2α-positive staining contributed more than any other clinicopathological factor to predicting the variation in the future length of survival. Analysis with luminal type breast cancer (n=59), in which the risk of recurrence is generally low and which is treated postoperatively with hormone therapy alone, showed that cancer recurrence and poor patient outcomes strongly associate with nucleolar CK2α-positive stain (n=7; p=0·037) by Kaplan-Meier analysis. In this case, nucleolar CK2α is considered a sole independent prognostic factor (HR 2·11 [95% CI 0·27-3·69]; p=0·0087). Evaluation of this method in other solid tumours is ongoing. Our results suggest that the evaluation of nucleolar CK2α-positive staining might be a new and independent prognostic factor that can improve treatment efficacy and patient quality of life after surgical resection of the tumour. The application of this analysis could contribute to early treatment strategy decisions and to companion diagnostics. The protein interactome of nuclear CK2 complexes showed CK2α's role in protein synthesis and RNA post-transcriptional modification. Specifically, a more detailed evaluation of CK2α function in nucleolar events in malignant cells might lead to the development of novel, more accurately targeted anti-cancer therapeutics. In conclusion, evaluating nucleolar CK2α staining in combination with other clinicopathological indicators might allow for a more accurate prediction of future recurrence or metastasis, which cannot be accomplished by current methods. Japan Agency for Medical Research and Development (grant number 20lm0203006j0004).

Component analysis of nucleolar protein compartments using Xenopus laevis oocytes.

The nucleolus is a multi‐compartment, non‐membrane‐bound organelle within the nucleus. Nucleolar assembly is influenced by proteins capable of phase separation. Xenopus laevis oocytes contain hundreds of large nucleoli that provide experimental access for nucleoli that is unavailable in other systems. Here we detail methods to streamline the in vivo analysis of the compartmentalization of nucleolar proteins that are suspected of phase separation. The nucleolus is the main hub of ribosome biogenesis and here we present data supporting the division of proteins into nucleolar domains based on their function in ribosome biogenesis. We also describe the use of vital dyes such as Hoechst 33342 and Thioflavin T in nucleolar staining. Additionally, we quantify nucleolar morphology changes induced by heat shock and actinomycin D treatments. We suggest these approaches will be valuable in a variety of studies that seek to better understand the nucleolus, particularly those regarding phase separation. These approaches may also be instructive for other studies on phase separation, especially in the nucleus.

Clinical Significance of Antinucleolar Antibodies: Biomarkers for Autoimmune Diseases, Malignancies, and others.

Nucleolar staining is one of the standard patterns in immunofluorescence antinuclear antibodies (ANA), seen in 5-9% of ANA in various conditions. Antinucleolar antibodies (ANoA) are classified into 3 patterns in the International Consensus on ANA Patterns (ICAP) classification; AC-8 homogeneous pattern, AC-9 clumpy pattern, and AC-10 punctate pattern. Specificities known to show AC-8 include anti-Th/To, -PM-Scl, -nucleophosmin/B23, -nucleolin/C23, -No55, and others. AC-9 is seen by anti-fibrillarin/U3RNP and AC-10 by anti-RNA polymerase I and hUBF/NOR-90. ANoA has been classically known to be associated with scleroderma (SSc) and the characterization of nucleolar antigens identified several autoantigens recognized by SSc autoantibodies. The clinical association of anti-Th/To, PM-Scl, fibrillarin/U3RNP, and RNA polymerase I with SSc or SSc-overlap syndrome is well established, and commercial assays are developed. Anti-hUBF/NOR90, nucleophosmin/B23, and nucleolin/C23 are known for decades and reported in systemic autoimmune rheumatic diseases (SARDs), malignancies, graft versus host disease (GVHD), and others; however, their clinical significance remains to be established.

Evaluation of antitumoral effect of mistletoe fruit extract on Ehrlich ascites tumor cells with muse cell analyzer and argyrophilic nucleolar organizer region staining method

Abstract Introduction Mistletoe has been used alone or as a complementary therapy in the treatment of different diseases for years. In this study, the antitumoral effect of mistletoe fruit extract on Ehrlich ascites tumor (EAT) cells was evaluated. Materials and Methods EAT cells from preformed stock mice were transferred to culture dishes containing 5-fluorouracil (5-FU) and mistletoe extracts at different doses (100, 200, 400, and 800 μg/ml). These cells were incubated at 37 °C in an environment with 95% humidity and 5% CO2. At the end of the incubations, the apoptosis status of the cells, cell cycle, mitochondrial membrane potential, and proliferation status with the argyrophilic (Ag) nucleolar organizer region staining (NORs) method were evaluated. Results As a result, it was observed that the mistletoe fruit extract and 5-FU induce apoptosis of EAT cells. It was concluded that the 5-FU substance arrests the cell cycle at the G0/G1 stage, while the mistletoe arrests the cell cycle at the S and G2/M stages. The depolarization rate of the mistletoe treated cells was higher. As a result of the evaluation made with the AgNORs method, it was seen that mistletoe and 5-FU could be effective in reducing the proliferation of EAT cells. Conclusions It was seen that mistletoe fruit extract could be effective in stimulating the apoptosis and depolarization of cancer cells. The results of other studies in the literature and our study support each other. It was concluded that the mistletoe plant may be useful in cancer treatment.

Avaliação quantitativa da expressão da proteína PCNA e das AgNORs em ameloblastomas

In order to assess the proliferative behaviour of the ameloblastoma epithelium nine cases of this neoplasm were retrieved from the files of the Oral Pathology Service laboratory of Rio Grande do Sul Federal University School of Dentistry. Two proliferation marker techniques were employed: the AgNor - argyrophilic nucleolar organizer regions staining and the proliferation cell nuclear antigen (PCNA) immunohistochemical assessment technique. Each case was submitted to a quantitative and comparative statistical analysis of the results obtained for the two techniques. The conclusion arrived at shows ameloblastoma presenting benign behavior, an average AgNOR index of 1.612, and a PCNA proteindependent proliferative activity with average immunoreactivity of 51.61%. The histological patterns analyses of the neoplastic epithelial component demonstrates that cells resembling pre-ameloblasts have obtained figures statistically higher to those obtained for cells resembling the stellate reticulum of the enamel organ, in both techniques employed. Therefore, it might be inferred that cells resembling pre-ameloblasts hold the proliferative potential of ameloblastomas and are probably responsible for the growth characteristics and infiltrative ability of this tumour.

Joint contractures responsive to immunosuppressive therapy in a girl with childhood\u2010onset systemic sclerosis double\u2010seropositive for rare anti\u2010nucleolar autoantibodies: a case report

BackgroundSystemic sclerosis (SSc; scleroderma) is an autoimmune connective tissue disease that affects the skin and subcutaneous tissue, in addition to the internal organs of the whole body. Onset in childhood is uncommon; however, both patients with childhood-onset and adult-onset SSc are positive for anti-nuclear antibodies (ANAs).Detection of SSc-related anti-nuclear antibodies is often useful for predicting clinical features, disease course, and outcomes.Case presentationA 5-year-old Japanese female manifested gradually progressive abnormal gait disturbance, regression of motor development, Raynaud’s phenomenon, and the shiny appearance of the skin of the face and extremities at age 2. On admission, she presented a mask-like appearance, loss of wrinkles and skin folds, puffy fingers, moderate diffuse scleroderma (18/51 of the modified Rodnan total skin thickness score), and contracture in the ankle and proximal interphalangeal joints. Grossly visible capillary hemorrhage on nail fold and severe abnormal capillaroscopy findings including bleeding, giant loop and disappearance of capillaryconsistent with the late phase in SSc. A skin biopsy showed fibrous thickening of the dermis, entrapment of an eccrine sweat glands, and thickened fiber. Chest high-resolution computed tomographic scanning demonstrated patchy areas of ill-defined air-space opacity and consolidation predominantly involving the posterior basilar aspects of the lower lobes presenting withinterstitial lung disease. Positive ANA (1:160 nucleolar and homogeneous nuclear staining by indirect fluorescent antibody technique) and double-seropositive for anti-Th/To and anti-PM-Scl antibodies were identified. She was diagnosed with diffuse cutaneous SSc based on the Pediatric Rheumatology European Society/American College of Rheumatology/European League Against Rheumatism Provisional Classification Criteria for Juvenile Systemic Sclerosis and was successfully treated with immunosuppressive agents, including methylprednisolone pulses and intravenous cyclophosphamide.ConclusionsWe experienced the first case of juvenile SSc with anti-PM-Scl and anti-Th/To antibodies. ILD was identified as a typical feature of patients with these autoantibodies; however, diffuse cutaneous SSc and joint contraction were uncharacteristically associated. The case showed unexpected clinical findings though the existence of SSc-related autoantibodies aids in determining possible organ involvement and to estimate the children’s outcome.

Intracellular localization of CK2α as a prognostic factor in invasive breast carcinomas.

Overexpression of the ubiquitous protein kinase, CK2α, has been reported in various human cancers. Here, we demonstrate that nuclear and nucleolar CK2α localization in invasive ductal carcinomas of the breast is a reliable predictor of poor prognosis. Cellular localization of CK2α in nuclei and nucleoli was analyzed immunohistochemically using surgical tissue blocks from 112 patients, who had undergone surgery without neoadjuvant chemotherapy. Clinical data collection and median follow‐up period were for more than 5 y. In total, 93.8% of patients demonstrated elevated CK2α expression in nuclei and 36.6% of them displayed elevated expression predominantly in nucleoli. Clinicopathological malignancy was strongly correlated with elevated nuclear and nucleolar CK2α expression. Recurrence‐free survival was significantly worse (P = .0002) in patients with positive nucleolar CK2α staining. The 5‐y survival rate decreased to a roughly 50% in nucleolar CK2α‐positive patients of triple‐negative (P = .0069) and p Stage 3 (P = .0073) groups. In contrast, no patients relapsed or died in the triple‐negative group who exhibited a lack of nucleolar CK2α staining. Evaluation of nucleolar CK2α staining showed a high secondary index with a hazard ratio of 6.629 (P = .001), following lymph node metastasis with a hazard ratio of 14.30 (P = .0008). Multivariate analysis demonstrated that nucleolar CK2α is an independent factor for recurrence‐free survival. Therefore, we propose that histochemical evaluation of nucleolar CK2α‐positive staining may be a new and robust prognostic indicator for patients who need further treatment. Functional consequences of nucleolar CK2 dysfunction may be a starting point to facilitate development of novel treatments for invasive breast carcinoma.

An extraordinary case of oral leukocytoclastic vasculitis demonstrating igd deposition around dermal vessels and within mucosal keratinocytes

Cutaneous vasculitides include a widespread and heterogeneous cluster of diseases affecting the blood vessels that are clinically characterized by polymorphic skin lesions, including palpable purpura, urticarial and/or necrotic-ulcerative lesions. Often, they can be manifestations of a systemic disease. Selected cases occur in the mouth. A 75-year-old female presented to her physician for the sudden appearance of blisters in her mouth, with severe orodynia and no history of other diseases or medication intake. A skin biopsy of the oral mucosa yielded a diagnosis of leukocytoclastic vasculitis. The direct immunofluorescence and immunohistochemistry stains demonstrated deposits of IgD, IgG, IgA, IgM, kappa, lambda, C1q, C3c, albumin and fibrinogen at the upper dermal neurovascular plexus. IgD also demonstrated positive nucleolar staining of the keratinocytes. Our case involves a rare presentation of oral cutaneous vasculitis with immune deposits of several immunoglobulins, complement, albumin and fibrinogen. Our case adds importance to studies of the IgD role in antigenic complex immune responses, especially in the mouth.

Live-cell imaging of the nucleolus and mapping mitochondrial viscosity with a dual function fluorescent probe.

Visualization of sub-cellular organelles allows the determination of various cellular processes and the underlying mechanisms. Herein, we report a fluorescent probe, bearing push-pull substituents emitting at 600 nm and its application in cellular imaging. The probe shows dual imaging of mitochondria and nucleoli and maps mitochondrial viscosity in live cells under various physiological variations and show minimum cytotoxicity. Nucleolar staining is confirmed by RNAase digestion.

The Correlate between Argyrophilic Nucleolar Organizer Region Stain and Ki-67 Immnochistochemistry in Diagnostic Breast Cancer

Introduction: This experimental study gauged the value of argyrophilic nucleolar organizer region (AgNOR) staining as a possible technique for the estimation of cell kinetics in conventional histology sections, in benign and malignant breast lesions. Methods: With a silver staining technique and immunohistochemistry, we associated the numbers of AgNORs and Ki67 scores in 30 breast carcinomas and 10 benign breast lesions. Results: The mean values of Ag NORs silver stain dots count for normal, benign, grade II and III were 1.28±0.17, 2.83±0.68, 5.23±0.87 and 7.32±0.92, respectively. There was a statistically significant difference (p≤0.001) was noticed between the all individual groups, among the normal and breast lesion as well as among the GII and GIII. Immunohistochemical Results of Ki-67 protein exhibited homogenous golden-brown color in control case and a positive brown granules or diffuse dark brown color in the nuclei of both benign and malignant cases under the 400X magnify examined under the light microscope. Discussion: AgNOR counts performed on routine formalin-fixed paraffin sections could provide substantial kinetic evidence. Additionally, the difference in AgNOR counts between benign and malignant tumors is such that they may be of diagnostic worth.

The Correlate between Argyrophilic Nucleolar Organizer Region Stain and Ki-67 Immnochistochemistry in Diagnostic Breast Cancer

The Correlate between Argyrophilic Nucleolar Organizer Region Stain and Ki-67 Immnochistochemistry in Diagnostic Breast Cancer

THU0600 A CASE OF SYSTEMIC SCLEROSIS COMPLICATED BY RENAL CRISIS: POTENTIAL ETIOPATHOGENETIC ROLE OF CYTOMEGALOVIRUS AND TREATMENT

Background:Scleroderma renal crisis (SRC) is a rare complication of systemic sclerosis (SSc), which can be triggered by viruses, such as Cytomegalovirus (CMV). SRC presents as a new-onset accelerated-phase hypertension with/without rapidly progressive renal failure.Objectives:Here we describe the case of a patient developing SSc complicated by the appearance of SRC after a recent episode of acute Cytomegalovirus infection.Methods:A 66-year-old male was referred to our Scleroderma Unit in March 2019. He presented with widespread skin rash, exertional dyspnoea and peripheral oedemas. He reported a myocarditis due to CMV occurred in October 2018. Antibodies anti-CMV IgM were detected in his serum. The patient developed a progressive cutaneous involvement characterized by diffuse oedema, sclerosis and melanoderma. Subsequently, Raynaud’s phenomenon, puffy hands and pitting scars occurred. Laboratory tests showed positive ANA in a titer of 1:640 in a nucleolar staining pattern. Additionally, persistence of anti-CMV IgM was found. Skin biopsy showed scleroderma-like finding. Nailfold capillaroscopy revealed a SSc pattern. Chest high resolution computed tomography displayed basal interstitial thickening and subpleuric ground-glass opacities. Therefore, the patient was diagnosed with SSc. Three weeks later he developed severe hypertension and a rapid, progressive renal impairment. Serum creatinine increased (up to 4.15 mg/dl), glomerular filtration rate impaired (25 ml/min). Renal biopsy (picture A, B) revealed acute thrombotic microangiopathy. A diagnosis of thrombotic thrombocytopenic purpura was excluded. The patient was diagnosed with SRC and we started therapy with ACE-inhibitor and loop diuretic. Even if the dosage of ACE-inhibitor was increased up to the maximum tolerate dose, his renal function did not improve and the blood pressure control was inadequate. Consequently, the patient underwent plasma exchange (PEx) sessions. Two weeks later there was an improvement of renal function and blood pressure normalized. Six months later the disease was controlled: glomerular filtration rate was 41 ml/min and blood pressure was within the normal range. The patient was treated with ACE-inhibitor and underwent fortnightly apheretic sessions. Treatment for scleroderma vasculopathy is ongoing.Results:Viral infections may be responsible for SSc. A brief interval between an acute viral infection and the onset of SSc may suggest CMV as a possible trigger for the disease. Similarly, other infectious agents could be involved in the multistep and multifactorial mechanism of SSc. This case sheds light on the potential and intriguing role of CMV in SSc. Moreover, it leads us to hypothesize a CMV possible direct role in sclerodermal kidney damage. Use of ACE-inhibitor significantly reduced the mortality rate due to this complication. Exact therapeutic mechanism of PEx in the treatment of SSc is unclear.Conclusion:In our case the integrated ACE-inhibitor-PEx approach has showed effectiveness and safety in the management of SRC.