Nucleolin and nucleophosmin expression patterns in pulmonary adenocarcinoma invading the pleura and in pleural malignant mesothelioma.

BACKGROUND: Visceral pleural invasion (VPI) in adenocarcinoma of the lung is considered a poor prognostic factor. The purpose of this study was to analyze nucleolin and nucleophosmin expression in pulmonary adenocarcinoma (PA) with VPI and in pleural malignant mesothelioma. METHODS: The study was conducted on the basis of 19 pathologically-confirmed cases of adenocarcinoma of the lung and 29 cases of epithelioid malignant mesothelioma. The nucleolin and nucleophosmin expression was assessed immunohistochemically and analyzed with image analysis software. RESULTS: Nucleolin expression was lower while nucleophosmin was higher in pleural invasion of pulmonary adenocarcinoma than in the central part of the tumor. Differences in subpopulations of cells with different expression of proteins studied were also found. Malignant mesothelioma showed lower nucleolin expression than adenocarcinoma of the lung but no differences in nucleophosmin expression were found. CONCLUSIONS: The results of our study suggested that lower nucleolin and higher nucleophosmin expression may be related to higher invasiveness of adenocarcinoma of the lung. Differences in nucleolin expression between pulmonary adenocarcinoma and malignant mesothelioma indicate another aspect of biology of these pleura-invading cancers that requires further study. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: Differences in nucleolin and nucleophosmin expression in pleura invading pulmonary adenocarcinoma indicate the involvement of these proteins in its locoregional spread while differences in nucleolin expression between pulmonary adenocarcinoma and malignant mesothelioma suggest another aspect of biology of these cancers. WHAT THIS STUDY ADDS: This is the first study on nucleolin and nucleophosmin expression in pleural malignant mesothelioma and pleura-invading pulmonary adenocarcinoma. Our findings may assist in understanding the mechanisms of locoregional spread of adenocarcinoma and differences between these two pleura-invading cancers.

Nucleolin and Nucleophosmin Expression in Gleason 3 and Gleason 4 Prostate Cancer With Seminal Vesicles Invasion (pT3b).

BACKGROUND/AIM: The aim of this study was to analyze the expression of nucleolin (NCL) and nucleophosmin (NPM) in prostate adenocarcinoma and in its loco-regional spread in the form of seminal vesicle invasion (SVI). MATERIALS AND METHODS: The study was performed on tissue microarrays of 40 cases of Gleason 3+4 pT3b prostate cancers including tissue samples from SVI. The expression of NCL and NPM was detected immunohistochemically and analyzed with image analysis software. RESULTS: The expression of NCL and NPM were higher in cancer cells within a prostate gland than in SVI. Gleason 4 pattern showed higher expression of NPM than Gleason 3 pattern cells. CONCLUSION: Differences in nuclear NCL and NPM expression in cancer cells between the prostate gland and SVI may indicate involvement of these proteins in loco-regional spread of adenocarcinoma of the prostate. Differences in NPM expression in Gleason 3 and Gleason 4 pattern suggest involvement of this protein in the differentiation of prostate cancer.

Nucleolin and nucleophosmin expression in seminomas and non-seminomatous testicular tumors.

INTRODUCTION: Testicular tumors are heterogeneous group of neoplasms divided mainly into two types: seminomas and non-seminomas. Nucleolin (NCL) and nucleophosmin (NPM) are abundant nucleolar proteins involved in many physiologic and pathologic processes including cancer. Their overexpression was found in many tumors but it was not studied in testicular cancer. MATERIAL AND METHODS: The study was performed on tissue microarrays of 19 seminomas, 21 embryonal carcinomas and 11 yolk sac tumors. The expression of NCL and NPM was detected with monoclonal antibodies and visualized with EnVision FLEX/HRP technique. Immunohistochemical reactions were measured with Aperio ImageScope Software and analyzed as means of percentages of all immunopositive cells in three groups of reaction intensity, i.e. 3+, 2+, and 1+ as well as of H-score. RESULTS: Seminomas showed higher expression of nucleolin indicated by higher H-score and higher percentage of positive cells than non-seminomas. The differences in subpopulations of NCL-positive cells were also found. Embryonal carcinomas and yolk sac tumors showed lower expression of NCL than seminomas indicated by H-score. The percentage of NCL-positive cells did not differ between embryonal carcinomas and seminomas while there were significant differences in subpopulations of cells. The percentage of NCL-positive cells in yolk sac tumors was lower than in seminomas. The results show different heterogeneity of subpopulations of NCL-positive cells in embryonal carcinomas and yolk sac tumors compared to seminomas. The analysis of nucleolin expression in embryonal carcinomas and yolk sac tumors showed no differences between these two tumor types. No differences in nucleophosmin expression between seminomas and non-seminomas were found. CONCLUSIONS: The differences in the expression of nucleolin between two groups of germ cell testicular tumors found in the current study indicate a new aspect of biology of these neoplasms and require further studies on the role of nucleolin in germ cell tumorigenesis.

Primary malignant melanoma of esophagus: clinicopathologic characterization of 20 cases including molecular genetic profiling of 15 tumors.

Primary malignant melanoma of esophagus is very rare, and its clinicopathologic and genetic features have not been extensively investigated. In this study, 20 tumors from 14 male and 6 female patients (40-79 years old) were evaluated. Dysphagia, chest pain, and weight loss were frequent symptoms. Thirteen melanomas, including two with multiple lesions, involved the distal third of esophagus. The median tumor diameter was 6 cm. Epithelioid morphology, moderate atypia, and pigmentation were typical findings. None of the patients had melanoma elsewhere, and all tumors exhibited a junctional peri-epithelial component consistent with a primary lesion. The median mitotic activity was 11 per 10 high-power fields (range, 0-31). Nine patients died of tumor within 4-22 months, however, two showed long-term (96 and 104 months) survival. In 15 cases, tissue for further immunohistochemical and molecular studies were available. BRAF, KIT, and NRAS mutation status was assessed by Sanger sequencing in all 15 tumors. The next-generation sequencing of 50 or 409 genes was performed in five and three cases, respectively. IGF1R expression indicating activation of the IGF axis was seen in 82% (9/11) of tumors. However, no BRAF mutations were identified. In 33% (5/15) of tumors, NRAS mutations were detected. KIT expression was seen in 50% (7/14) of melanomas including single KIT mutant. Two of three tumors evaluated with 409 genes panel revealed multiple driver mutations indicating sub-clonal expansion, whereas a single mutation (TSC1 p.H371Q) was the sole change in the third case. SF3B1 p.K666T and p.R625C mutations were detected in two cases. However, no co-occurrence of SF3B1 and GNAQ or GNA11 mutations, seen in uveal melanoma, was detected. FBXW7 p.R465C and p.R479G mutations, linked to cancer progression, were found in two of eight tumors. In summary, esophageal melanoma mutation profile indicates complexity of molecular mechanisms underlying its pathogenesis.

Assessment of morphological changes and steroid receptors in the uteri of postmenopausal women.

INTRODUCTION: The morphology of the endometrium constantly changes in the reproductive period, depending on the levels of ovarian steroid hormones, and undergoes atrophic changes during menopause as a result of their insufficiency. The purpose of this study was to analyze morphological and morphometric changes in the mucous and muscle layers of the uterine wall in postmenopausal women, and to assess localization and number of cells showing the expression of steroid hormone receptors, namely estrogen receptor α (ER-α), progesterone receptor (PR), and androgen receptor (AR) in glandular epithelial cells and smooth muscle cells in particular groups of women. MATERIAL AND METHODS: The study material consisted of uterine specimens sectioned across the full thickness of the uterine wall, and embedded in 164 paraffin blocks. The specimens came from women without menopausal hormone therapy (MHT) operated due to reproductive organ prolapse or uterine myomas. The material was divided into four groups depending on the time interval from menopause to surgery: group I - from 1 to 5 years after menopause, group II - from 6 to 10 years after menopause, group III - more than 11 years after menopause, and group IV - women over 70 years of age. The sections were stained by standard HE, Masson's trichrome, and immunohistochemical methods (ERα, PR, AR). Quantitative assessment of the results was based on computer image analysis. RESULTS: Analysis of morphological changes in the endometrium and myometrium revealed the presence of increasing regressive changes, such as various types of atrophy, fibrosis, and calcification, augmented over time from the last menstruation. Furthermore, endometrial polyps, foci of endometriosis, and leiomyomas were observed. Based on the results of morphometric measurements, a constant decrease in the endometrial and myometrial thickness was noticed in the studied groups (I-IV). Significant differences between the groups were observed in the number of ER-α positive cells in the myometrium, but not in the endometrial glandular epithelium. Statistically significant differences in the number of AR positive cells were detected in the endometrial epithelium and in the uterine muscle. The analysis the number of PR positive cells demonstrated differences between the groups in the endometrial stroma and the myometrium. CONCLUSION: The uterus of postmenopausal woman undergo major morphological changes (mainly atrophic lesions in the endometrium and myometrium), leading to a decline in their morphometric parameters over time from the last menstruation. Localization and number of cells showing the expression of steroid receptors: ER-α, PR, and AR in the uterus of postmenopausal women, depending on the time interval from the last menstruation.

Wegener's granulomatosis and pyoderma gangrenosum--rare causes of facial ulcerations.

BACKGROUND: Pyoderma gangrenosum (PG) is caused by immune system dysfunction, and particularly improper functioning of neutrophils. At least half of all PG patients also suffer from autoimmunological diseases, one of which is Wegener granulomatosis (WG). The purpose of this article was to compare cases of patients with WG and PG in terms of their clinical course, histopathology, and applied treatment. In both, histopathological features are not fully distinct. Data from microbiological and immunological evaluation and clinical presentation are required to establish the diagnosis. We also present the case of a patient with WG and deep facial skin lesions not responding to standard treatment. METHODS: Systematic review of the literature in PubMed using the search terms "Wegener granulomatosis AND Pyoderma gangrenosum" and case report. RESULTS: The finding of 22 reports in the literature (PubMed) suggests that it is a rare phenomenon. This study revealed a similar rate of comorbidity of WG and PG in both genders and an increased incidence of both diseases after the age of 50. Among skin lesions there was a dominance of ulceration, most often deep and painful, covering a large area with the presence of advanced necrosis and destruction of the surrounding tissue. The most common location proved to be the cervical-cephalic area. The most popular treatment included steroids with cyclophosphamide. DISCUSSION: The rarity of the coexistence of these two diseases results in a lack of effective therapy. In such cases sulfone derivatives are still effective and provide an alternative to standard immunosuppression methods. Hyperbaric therapy and plasmapheresis can also play an important complementary role.

Effect of methylprednisolone, hyaluronic acid and pioglitazone on histological remodeling of temporomandibular joint cartilage in rabbits affected by drug-induced osteoarthritis.

BACKGROUND: The aims of this study were to assess the anti-degenerative effects of pioglitazone and to compare these effects with those of methylprednisolone and hyaluronic acid on drug-induced osteoarthritis in rabbits' temporomandibular joint cartilage. MATERIAL AND METHODS: The experiment was conducted on 40 Californian white rabbits. Degenerative changes were induced by intra-articular injections of papain. Subsequently, all of the animals were randomly assigned to one of four groups: 1) a control group that received no medications; 2) a group treated with 4 intra-articular injections of 2 mg (0.2 ml) of hyaluronic acid at weekly intervals; 3) a group treated with 4 intra-articular injections of 2 mg (0.1 ml) of methylprednisolone at weekly intervals; 4) a group administered pioglitazone orally in daily doses of 2 mg/kg of body weight. Four weeks after the beginning of drug administration, the rabbits were sacrificed. Sagittal sections of the intra-articular cartilage (discs) and mandibular condyles were stained with hematoxylin and eosin by the PAS technique and with van Gieson's solution. Histologic examinations, as well as cartilage thickness and number of cell layers measurements, were performed. RESULTS: Histologic assessment in cases of arthritis-associated pathologies revealed that changes occurred most frequently in the control group and least frequently in the pioglitazone group. There were no differences in the histological structures of the intra-articular discs. Cartilage thickness measurements demonstrated the thinnest cartilage in group 2 and the thickest in group 3. Analysis of cell layer numbers showed the most numerous layers in the pioglitazone group and the least in the control group. CONCLUSION: Pioglitazone and hyaluronic acid showed anti-degenerative properties compared to methylprednisolone in an animal model.

Effects of immunosuppressive treatment on protein expression in rat kidney.

The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents' toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins' synthesis. Very slight differences were observed between the group receiving cyclosporine, mycophenolate mofetil, and glucocorticoids (CMG) and the control group. In contrast, compared to the control group, animals receiving tacrolimus, mycophenolate mofetil, and glucocorticoids (TMG) exhibited higher expression of proteins responsible for renal drug metabolism and lower expression levels of cytoplasmic actin and the major urinary protein. In the TMG group, we observed higher expression of proteins responsible for drug metabolism and a decrease in the expression of respiratory chain enzymes (thioredoxin-2) and markers of distal renal tubular damage (heart fatty acid-binding protein) compared to expression in the CMG group. The consequences of the reported changes in protein expression require further study.

The CXCR7 chemokine receptor promotes B-cell retention in the splenic marginal zone and serves as a sink for CXCL12.

The splenic marginal zone (MZ) is comprised of specialized populations of B cells, dendritic cells, and macrophages that are uniquely arrayed outside the white pulp follicles to screen the blood for bacterial and other particulate Ags. Mechanisms responsible for MZ B-cell formation, localization, retention, and function are understood to include antigenic specificity, transcription factors, integrins, and surface receptors for soluble ligands such as S1P. Here, we add to this repertoire by demonstrating that the receptor for CXCL12, CXCR7, is expressed on MZ but not on follicular B cells. Treatment of mice with CXCR7 inhibitors led to disruption of MZ architecture, reduced numbers of MZ B cells, and altered granulocyte homeostasis associated with increasing serum levels of CXCL12. CXCR7 thus appears to function as a scavenger receptor for CXCL12 on MZ B cells.

Characterization of monoclonal antibodies to the plasma cell alloantigen ENPP1.

The ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) has documented roles in mineralization, nucleotide recycling, and insulin resistance. While ENPP1 was first identified as an alloantigen on mouse plasma cells (PCs), later studies revealed expression in many tissues. Previously described monoclonal antibodies against ENPP1 expressed at the cell surface recognized cells only from mice bearing the a allotype, ENPP1(a), precluding studies of mice bearing the alternative allele, ENPP1(b). Here, we characterize a novel anti-ENPP1 monoclonal antibody that recognizes both alleles and can be used for flow cytometry.

IFN regulatory factor 8 restricts the size of the marginal zone and follicular B cell pools.

Transcriptional control of marginal zone (MZ) and follicular (FO) B cell development remains incompletely understood. The transcription factor, IFN regulatory factor (IRF)8, is known to play important roles in the differentiation of early B cells. In this article, we demonstrate that IRF8 is also required for normal development of MZ and FO B cells. Mice with a conventional knockout of Irf8 (IRF8(-/-)) or a point mutation in the IRF association domain of IRF8 had increased numbers of MZ B cells. To determine the B cell-intrinsic effects of IRF8 deficiency, we generated mice with a conditional allele of Irf8 crossed with CD19-Cre mice (designated IRF8-conditional knockout [CKO]). These mice had enlarged MZ and increased numbers of MZ and FO B cells compared with controls. The FO B cells of CKO mice exhibited reduced expression of CD23 and moderately increased expression of CD21. Gene-expression profiling showed that increased B cell production in IRF8-CKO mice was associated with changes in expression of genes involved in regulation of transcription, signaling, and inflammation. Functional studies showed that IRF8-CKO mice generated normal Ab responses to T-independent and T-dependent Ags. Thus, IRF8 controls the expansion and maturation of MZ and FO B cells but has little effect on B cell function.

Clinical evidence that very small embryonic-like stem cells are mobilized into peripheral blood in patients after stroke.

BACKGROUND AND PURPOSE: In a murine model of stroke, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult murine bone marrow that could be mobilized into peripheral blood (PB). This raised the question of whether a similar population of cells is mobilized in human stroke patients. METHODS: We evaluated a number of cells that corresponded to VSEL SCs in the PB of 44 stroke patients and 22 age-matched controls. After each patient's stroke, PB samples were harvested during the first 24 hours, on day +3, and on day +7 and then compared with normal controls. The circulating human cells with the phenotype of VSEL SCs were evaluated in PB by real-time quantitative polymerase chain reaction, fluorescence-activated cell sorting analysis, and direct immunofluorescence staining. In parallel, we also measured the serum concentration of stromal derived factor-1 by ELISA. RESULTS: In stroke patients, we found an increase in the number of circulating cells expressing SC-associated antigens, such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating primitive cells expressing the VSEL phenotype (CXCR4(+)lin(-)CD45(-) small cells), mRNA for Octamer-4 and Nanog, and Octamer-4 protein. All changes were accompanied by an increased serum concentration of stromal derived factor-1. Additionally, we found a positive correlation between stroke extensiveness, stromal derived factor-1 concentration in serum, and the number of CXCR4(+) VSEL SCs circulating in the PB. CONCLUSIONS: We conclude that stroke triggers the mobilization of CXCR4(+) VSEL SCs that have potential prognostic value in stroke patients. However, the potential role of these mobilized cells in brain regeneration requires further study.

The expression and intranuclear distribution of nucleolin in HL-60 and K-562 cells after repeated, short-term exposition to rotating magnetic fields.

PURPOSE: The aim of the study was to analyze the influence of rotating magnetic fields (RMF) on the expression and intranuclear distribution of nucleolin, protein involved in ribosome biosynthesis, in HL-60 (acute promyelocytic leukemia) and K-562 (chronic myelogenous leukemia) established human cell lines. MATERIALS AND METHODS: Cells were exposed to RMF for two chosen states of the magnetic field induction: B=10 mT and B=20 mT in experimental set-up for 30 min with 24-h intervals for four days. Cytospin slides were prepared and expression of nucleolin was detected using monoclonal antibodies. Parameters of fluorescence related to nucleolin were measured in at least 2000 tumor cells in each slide by a laser scanning cytometer with an argon laser. Percentages of cells in different phases of cell cycle were also analyzed. RESULTS: The repeated exposition of cells to RMF caused significant increase in nucleolin expression in the whole nucleus and in the nucleolin aggregates (NUA). The redistribution of nucleolin measured by changes in number of NUA was also observed. The exposition of both cell lines studied to RMF did not alter the cell cycle. CONCLUSION: The nucleolin is responsive to RMF in HL-60 and K-562. The increase of its expression may indicate a reaction of cells to RMF and it may influence their other biological properties.

Intranuclear nucleolin distribution during cell cycle progression in human invasive ductal breast carcinomas in relation to estrogen receptor status.

BACKGROUND: Nucleolin (NU) can be found in the nucleus in aggregates and in a diffused form. The expression and distribution of these two intranuclear pools of nucleolin was compared in estrogen receptor (ER)-positive and -negative invasive ductal breast carcinomas in relation to cell cycle phases. MATERIALS AND METHODS: Using a laser scanning cytometer NU was measured within the whole nucleus, within nucleolin aggregates (NUA) and within the NUA-free karyoplasm. The G1-, S- and G2M-phase cell subpopulations were distinguished on DNA histograms. RESULTS: The nuclei of ER-positive and ER-negative breast carcinomas in the G2M-phase differed in the expression of NU in the NUA and in the NUA-free karyoplasm, whereas in the S-phase they only differed in the NUA-free karyoplasm expression. In ER-positive carcinomas NU levels rose from G1 to G2M in both the NUA and the NUA-free karyoplasm, whereas in ER-negative carcinomas the NU levels increased only between G1/S in the NUA and between S/G2M in the NUA-free karyoplasm. CONCLUSION: There are significant differences in nucleolin expression and localization during cell cycle progression depending on ER status.

Peripheral blood lymphocytes P-glycoprotein (P-gp, gp-170) expression in allogenic kidney transplant patients.

AIM AND METHODS: P-glycoprotein (gp-170, P-gp) is a transmembrane transporter involved in drug, for example cyclosporine A, efflux from the cells thus limiting their intracellular concentration. Expression of the transporter on the surface of immune competent cells may be associated with poor prognosis in kidney transplant patients. The aim of the present study was to evaluate P-gp expression on the surface of CD4(+), CD8(+), CD19(+) and CD56(+) cells in kidney transplant patients treated with cyclosporine A as a main immunosuppressant, using flow cytometry. RESULTS: It was found that P-gp expression in kidney transplant patients with acute rejection did not differ significantly from transplanted patients without rejection studied in the same period after transplantation, as well as from the healthy controls. Administration of 3-day course of 1,000 mg/24 h methylprednisolone did not affect the expression of P-gp in the studied cells, except for significant elevation in CD56(+) cells, which disappeared at 2 weeks after cessation of steroid administration. CONCLUSION: Based on the results from the present study it can be concluded that P-gp expression is not a prognostic factor of acute kidney graft rejection.

[Expression and intranuclear distribution of nucleolin in estrogen receptor-negative and estrogen receptor-positive breast cancers in women measured by laser scanning cytometry]. / Ocena ekspresji nukleoliny i jej wewnatrzjadrowej dystrybucji w estrogeno-ujemnych i estrogeno-dodatnich rakach sutka u kobiet za pomoca laserowego cytometru skaningowego.

INTRODUCTION: Nucleolin (NU) is one of the most abundant nucleolar proteins. Nucleolin is mainly involved in ribosome biogenesis that is supported by the ability of NU to bind rDNA and modify the structure of chromatin by binding to histon H1. Estrogen receptor alpha (ERalpha) is a DNA-binding transcriptional factor. It is estimated that 69-85% of breast cancers in women are ERalpha-positive. The aim of the study was to assess the expression and intranuclear distribution of NU in invasive ductal and lobular breast cancers in women and their relationship to ERalpha-status, histologic type and grade of breast cancer, and lymph node status. For this purpose, laser scanning cytometry (LSC) was used. MATERIAL AND METHODS: Measurements were done in cytospins of cancer cells of 87 ductal and 11 lobular invasive breast cancers. The cells were labeled with mouse anti-human NU antibody followed by F(ab')2 fragments of FITC-conjugated goat anti-mouse antibody. Nuclei were counterstained with 5 microg/mL of propidium iodide in the presence of 100 microg/mL of RNase A. All measurements were performed using LSC. The following parameters of individual cancer cells were calculated: NU fluorescence within the nucleus, within nucleolin aggregates (NUA) and in the remaining karyoplasm, number of NUA, area of the nucleus and NUA. The percentage of ER-positive breast cancer cells was calculated in parallel by the automated image analysis in formalin-fixed sections using immunohistochemistry with anti-ERalpha antibody. The cut-off value for ER-negative tumors was set at 10% of positively stained nuclei. Statistical analysis was done using the Statistica 5.0 software. P values less than 0.05 were considered statistically significant. RESULTS: The mean area of the nucleus of ductal cancer cells was significantly higher and NU expression lower in ERalpha-negative cancers than in ERalpha-positive ones (p = 0.007 and p = 0.04, respectively). The mean area of NUA and NU expression in ductal cancers were higher than in lobular cancers (p = 0.03 and p = 0.02, respectively). The expression of NU within the nucleus and within the karyoplasm besides NUA was significantly higher in ductal than in lobular cancers (p = 0.02 and p = 0.04, respectively). The expression of NU in the remaining karioplasm of tumor cells of lymph node-positive cancers was lower than in node-negative ones (p = 0.04). The same relation was found for ductal cancers (p = 0.02). CONCLUSIONS: The differences in nucleolin expression and its intranuclear distribution in ERalpha-negative and ERalpha-positive breast cancers, as well as ductal and lobular cancers point to biologic differences between these carcinomas. The method used in the study may be applied to measurements of expression and intranuclear distribution of other nuclear proteins or to simultaneous measurement of expression and distribution of nuclear and cytoplasmic proteins.

Simultaneous measurement of nucleolin and estrogen receptor in breast cancer cells by laser scanning cytometry.

BACKGROUND: The purpose of the study was to test the feasibility of laser scanning cytometry (LSC) to simultaneously measure estrogen receptor (ER) and nucleolin (NU) expression in the nuclei of the same individual breast cancer cells. MATERIALS AND METHODS: Cancer cells from 64 breast tumors were labeled with anti-NU and biotinylated anti-ER antibodies, then with secondary FITC-conjugated antibody and streptavidin-APC conjugate, respectively, and measured by LSC. The expression of NU in the nucleus and NU aggregates (NUA), number of NUA, nuclear and NUA areas and ER expression were assessed for aspiration each cell. RESULTS: ER-bound APC fluorescence correlated with nuclear NU (r=0.65; p<0.001) and NUA-bound FITC fluorescence (r=0.59; p<0.001). Good correlation was found between percentages of ER-positive cells in LSC and by image analysis in paraffin-embedded sections (r=0.59, p<0.001). CONCLUSION: ER and NU expression can be measured simultaneously in the same nuclei of breast cancer cells.

Therapy with infliximab decreases the CD4+CD28- T cell compartment in peripheral blood in patients with rheumatoid arthritis.

Chronic inflammatory syndromes such as rheumatoid arthritis (RA) are associated with high frequencies of CD4+CD28- T cells. The number of these cells is genetically determined and may also be a consequence of chronic exposure to tumor necrosis factor-alpha (TNFalpha). The aim of this study was to examine whether the reported efficacy of anti-TNFalpha therapy in RA involves a resurgence of T cell populations that re-express CD28. After 36-week therapy with infliximab, a significant decrease in CD4+CD28- T cells in RA patients was observed in comparison with baseline. The results suggest that TNFalpha-neutralizing therapy may restore T cell homeostasis and reduce expansion of the CD28- T cells, which are cytotoxic and may contribute to organ manifestations in RA.

The increased number of CD4+CD28- T cells in patients after liver transplantation.

It has been observed that the organ manifestations in the patients with autoimmune diseases resulted from vessels damage may be caused by CD4+CD28- T lymphocytes, which express high levels of IFN gamma and posses cytolytic activity. The increased number of these cells in the recipients of allogenic bone marrow grafts and kidney grafts was also observed. The aim of the study was to evaluate the amount of CD4+CD28- T lymphocytes in the recipients of allogenic liver grafts. The size of CD4+CD28- T cells compartment has been determined in 20 patients after liver transplantation and 20 healthy subjects as control group using two-color FACS analysis. The frequency of CD4+CD28- T cells in the liver transplant recipient was 5.65% and was significantly higher than in control group 1.4% (p < 0.001). The increased level of cytotoxic CD4+CD28- T cells may be involved in vessels damage and gradual loss of graft function.

The expansion of CD4+CD28- T cells in patients with rheumatoid arthritis.

Clonal expansion of CD4+CD28- T cells is a characteristic finding in patients with rheumatoid arthritis (RA). Expanded CD4+ clonotypes are present in the peripheral blood, infiltrate into the joints, and persist for years. CD4+CD28- T cells are oligoclonal lymphocytes that are rare in healthy individuals but are found in high percentages in patients with chronic inflammatory diseases. The size of the peripheral blood CD4+CD28- T-cell compartment was determined in 42 patients with RA and 24 healthy subjects by two-color FACS analysis. The frequency of CD4+CD28- T cells was significantly higher in RA patients than in healthy subjects. Additionally, the number of these cells was significantly higher in patients with extra-articular manifestations and advanced joint destruction than in patients with limited joint manifestations. The results suggest that the frequency of CD4+CD28- T cells may be a marker correlating with extra-articular manifestations and joint involvement.