The Ruthenium Complex cis-(Dichloro)tetraammineruthenium(III) Chloride Presents Selective Cytotoxicity Against Murine B Cell Lymphoma (A-20), Murine Ascitic Sarcoma 180 (S-180), Human Breast Adenocarcinoma (SK-BR-3), and Human T Cell Leukemia (Jurkat) Tumor Cell Lines

The aim of present study was to verify the in vitro antitumor activity of a ruthenium complex, cis-(dichloro)tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) toward different tumor cell lines. The antitumor studies showed that ruthenium(III) complex presents a relevant cytotoxic activity against murine B cell lymphoma (A-20), murine ascitic sarcoma 180 (S-180), human breast adenocarcinoma (SK-BR-3), and human T cell leukemia (Jurkat) cell lines and a very low cytotoxicity toward human peripheral blood mononuclear cells. The ruthenium(III) complex decreased the fraction of tumor cells in G0/G1 and/or G2-M phases, indicating that this compound may act on resting/early entering G0/G1 cells and/or precycling G2-M cells. The cytotoxic activity of a high concentration (2 mg mL(-1)) of cis-[RuCl(2)(NH(3))(4)]Cl toward Jurkat cells correlated with an increased number of annexin V-positive cells and also the presence of DNA fragmentation, suggesting that this compound induces apoptosis in tumor cells. The development of new antineoplastic medications demands adequate knowledge in order to avoid inefficient or toxic treatments. Thus, a mechanistic understanding of how metal complexes achieve their activities is crucial to their clinical success and to the rational design of new compounds with improved potency.

Galectin-3 regulates peritoneal B1-cell differentiation into plasma cells

Extracellular galectin-3 participates in the control of B2 lymphocyte migration and adhesion and of their differentiation into plasma cells. Here, we analyzed the role of galectin-3 in B1-cell physiology and the balance between B1a and B1b lymphocytes in the peritoneal cavity. In galectin-3(-/-) mice, the total number of B1a lymphocytes was lower, while B1b lymphocyte number was higher as compared to wild-type mice. The differentiation of B1a cells into plasma cells was associated with their abnormal adhesion and location on the mesentery. The B220 and CD43, constitutively expressed by B1 lymphocytes, were respectively up- and downregulated in galectin-3(-/-) mice. Mononuclear cells were strongly adhered to the mesenteric membranes of both CD43(-/-) and galectin-3(-/-) mice, but in contrast to CD43(-/-) mice, the accumulation of B1 cells in peritoneal membranes in galectin-3(-/-) mice was accompanied by their functional differentiation into plasma cells. We have shown that in the absence of galectin-3, B1-cell differentiation into plasma cells is favored and the dynamic equilibrium of B1-cell populations in the peritoneum is maintained through a compensatory increase in B1b lymphocytes.

Lack of Galectin-3 Disturbs Mesenteric Lymph Node Homeostasis and B Cell Niches in the Course of Schistosoma mansoni Infection

Galectin-3 is a beta-galactoside-binding protein that has been shown to regulate pathophysiological processes, including cellular activation, differentiation and apoptosis. Recently, we showed that galectin-3 acts as a potent inhibitor of B cell differentiation into plasma cells. Here, we have investigated whether galectin-3 interferes with the lymphoid organization of B cell compartments in mesenteric lymph nodes (MLNs) during chronic schistosomiasis, using WT and galectin-3(-/-) mice. Schistosoma mansoni synthesizes GalNAc beta 1-4(Fuc alpha 1-3) GlcNAc(Lac-DiNAc) structures (N-acetylgalactosamine beta 1-4 N-acetylglucosamine), which are known to interact with galectin-3 and elicit an intense humoral response. Antigens derived from the eggs and adult worms are continuously drained to MLNs and induce a polyclonal B cell activation. In the present work, we observed that chronically-infected galectin-3(-/-) mice exhibited a significant reduced amount of macrophages and B lymphocytes followed by drastic histological changes in B lymphocyte and plasma cell niches in the MLNs. The lack of galectin-3 favored an increase in the lymphoid follicle number, but made follicular cells more susceptible to apoptotic stimuli. There were an excessive quantity of apoptotic bodies, higher number of annexin V(+)/PI(-) cells, and reduced clearance of follicular apoptotic cells in the course of schistosomiasis. Here, we observed that galectin-3 was expressed in nonlymphoid follicular cells and its absence was associated with severe damage to tissue architecture. Thus, we convey new information on the role of galectin-3 in regulation of histological events associated with B lymphocyte and plasma cell niches, apoptosis, phagocytosis and cell cycle properties in the MLNs of mice challenged with S. mansoni.

Relationship between plasma cells and hepatic stellate cells in autoimmune hepatitis

Autoimmune hepatitis is an inflammatory chronic disease of the liver, which frequently results in cirrhosis. The present study aimed to verify the relationship between plasma cells and stellate cells in autoimmune hepatitis. Thirty-three pre-treatment, 11 post-treatment, and 10 normal liver biopsies were reviewed. Sirius Red staining (for semi-quantitative analysis of hepatic fibrosis) and immunohistochemistry were carried out: double staining for smooth muscle alpha-actin and plasma cell marker (for detection and localization of activated hepatic stellate cells and plasma cells, respectively); and single staining for glial fibrillary acid protein (for detection of hepatic stellate cells). We found an increase in the stellate cell population, mainly with an activated phenotype in autoimmune hepatitis, compared to the control group (liver specimens with no histological evidence of liver disease, obtained from patients undergoing hepatic resection for benign liver mass). A positive significant correlation was observed between stellate cells and scores of fibrosis (measured by Sirius Red) and the number of plasma cells. Additionally, there was a co-localization of plasma cells and activated stellate cells. We also observed a reduction in the number of plasma cells, hepatic stellate cells, and fibrosis in patients who had successfully been treated and had a second liver biopsy post-treatment. Our findings support that the number of plasma cells can be a surrogate marker for the severity of liver disease, reflecting the number of hepatic stellate cells and the amount of fibrosis. It remains to be seen if this is a result of a direct interaction between the plasma cells and hepatic stellate cells or the response to the same stimulus that affects both cellular types. (c) 2010 Elsevier GmbH. All rights reserved.

Number of expressed cancer/testis antigens identifies focal adhesion pathway genes as possible targets for multiple myeloma therapy

Considering that the importance of cancer/testis (CT) antigens in multiple myeloma (MM) biology is still under investigation, the present study aimed to: (1) identify genes differentially expressed in MM using microarray analysis of plasma cell samples, separated according to the number of expressed CTs; (2) examine possible pathways related to MM pathogenesis; (3) validate the expression of candidate genes by quantitative real-time PCR (RQ-PCR). Three samples predominantly positive (>6 expressed), including the U266 cell line, and three samples predominantly negative (0 or 1 expressed CT for the 13 analyzed CT antigens), were submitted for microarray analysis. Validation by RQ-PCR from 24 MM samples showed that the ITGAS gene was downregulated in predominantly positive (>6 expressed CTs, p = 0.0030) and in tumor versus normal plasma cells (p = 0.0182). The RhoD gene was overexpressed in tumor plasma cells when compared to normal plasma cells (p = 0.0339). Results of the microarray analysis corroborate the hypothesis that MM could be separated into predominantly positive and predominantly negative expression. The differential expression of ITGA5 and RhoD suggests disruption of the focal adhesion pathway in MM and offers a new target field to be explored in this disease.

Frequency and prognostic relevance of cancer testis antigen 45 expression in multiple myeloma

Objective. This study aims to analyze the expression of cancer testis antigen 45 (CT45) in normal tissues and in plasma cell disorders and to identify possible associations with clinical data and prognosis in multiple myeloma (MM) patients. Materials and Methods. Expression of CT45 was studied in 20 normal tissues (testis, placenta, skeletal muscle, bladder, lung, spleen, heart, brain and fetal brain, thymus, uterus, stomach, mammary gland, pancreas, prostate, small intestine, kidney, adrenal gland, spinal cord, colon, and one pool of 10 normal bone marrow samples) and bone marrow aspirates from 3 monoclonal gammopathies of undetermined significance, 5 solitary plasmacytomas, 61 newly diagnosed MM patients and MM cell line U266 by reverse transcriptase polymerase chain reaction. Results. CT45 was positive in 3 of 20 (15%) normal tissues tested: lung, brain (both fetal and adult), and spinal cord. Among monoclonal gammopathies, CT45 was positive in 2 of 5 (40%) solitary plasmacytomas bone marrow aspirates, 10 of 61 (16%) MM bone marrow aspirates, and in the U266 MM cell line. Conclusions. We did not find associations between bone marrow histology and CT45 expression. However, we demonstrated for the first time that positive expression of CT45 was associated with poor prognostic (international Staging System) and poor outcomes in MM patients, meaning that CT45-positive cases presented seven times more chance of worse evolution than the negative ones. (C) 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Peroxiredoxin I is differentially expressed in multiple myelomas and in plasmablastic lymphomas

BACKGROUND: Plasmablastic lymphoma (PBL) and multiple myeloma (MM) are B cell-derived malignancies that share many morphologic and immunophenotypic traits, making the differential diagnosis particularly complicated. We have recently demonstrated that peroxiredoxin I (PrdxI) is expressed in plasma cells but not in B lymphocytes, suggesting that its expression is development-associated. AIM: To analyze PrdxI expression in PBL and in MM in order to study its utilization as an additional diagnostic molecular tool. METHODS AND RESULTS: Eight cases of PBL and nine of MM were studied by immunohistochemistry. We have demonstrated that PrdxI expression is closely connected with the immunoglobulin production capacity of the cells, which means high in MM, but absent in PBL cases, except one, wherein few cells were stained. CONCLUSIONS: We hypothesize PrdxI as a component of the unfolded protein response (UPR), an adaptive pathway essential for plasma cell differentiation. As we have not detected immunoglobulin in our PBL cases, we suggest that UPR was not activated in the cells, accounting for the impediment of the developmental process, and for the inhibition of PrdxI expression observed. PrdxI could be considered an additional plasma cell functional marker and could also be speculated as a therapeutic target in the treatment of MM. Oral Diseases (2008) 14, 741-746

Slower rescue of ER homeostasis by the unfolded protein response pathway associated with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and recurrent infections. Herein we addressed the role of unfolded protein response (UPR) in the pathogenesis of the disease. Augmented unspliced X-box binding protein 1 (XBP-1) mRNA concurrent with co-localization of IgM and BiP/GRP78 were found in one CVID patient. At confocal microscopy analysis this patient`s cells were enlarged and failed to present the typical surface distribution of IgM, which accumulated within an abnormally expanded endoplasmic reticulum. Sequencing did not reveal any mutation on XBP-1, neither on IRE-1 alpha that could potentially prevent the splicing to occur. Analysis of spliced XBP-1, IRE-1 alpha and BiP messages after LPS or Brefeldin A treatment showed that, unlike healthy controls that respond to these endoplasmic reticulum (ER) stressors by presenting waves of transcription of these three genes, this patient`s cells presented lower rates of transcription, not reaching the same level of response of healthy subjects even after 48 h of ER stress. Treatment with DMSO rescued IgM and IgG secretion as well as the expression of spliced XBP-1. Our findings associate diminished splicing of XBP-1 mRNA with accumulation of IgM within the ER and lower rates of chaperone transcription, therefore providing a mechanism to explain the observed hypogammaglobulinemia. (C) 2008 Elsevier Ltd. All rights reserved.

Osteoblastlike Cell Adhesion on Titanium Surfaces Modified by Plasma Nitriding

Purpose: The aim of this study was to evaluate the characteristics of various titanium surfaces modified by cold plasma nitriding in terms of adhesion and proliferation of rat osteoblastlike cells. Materials and Methods: Samples of grade 2 titanium were subjected to three different surface modification processes: polishing, nit riding by plasma direct current, and nitriding by cathodic cage discharge. To evaluate the effect of the surface treatment on the cellular response, the adhesion and proliferation of osteoblastlike cells (MC3T3) were quantified and the results were analyzed by Kruskal-Wallis and Friedman statistical tests. Cellular morphology was observed by scanning electron microscopy. Results: There was more MC3T3 cell attachment on the rougher surfaces produced by cathodic cage discharge compared with polished samples (P < .05). Conclusions: Plasma nitriding improves titanium surface roughness and wettability, leading to osteoblastlike cell adhesion. INT J ORAL MAXILLOFAC IMPLANTS 2011;26:237-244

Systemic chemotherapy-induced microsatellite instability in the mononuclear cell fraction of women with breast cancer can be reproduced in vitro and abrogated by amifostine

Objectives Microsatellite instability (MSI) induction by alkylating agent-based chemotherapy (ACHT) may underlie both tumor resistance to chemotherapy and secondary leukaemias in cancer patients. We investigated if ACHT could induce MSI in tumor-derived plasma-circulating DNA (pfDNA) and in normal peripheral blood mononuclear (PBMN) cells. We also evaluated if amifostine could interfere with this process in an in-vitro model. Methods MSI was determined in pfDNA, PBMN cells and urine cell-free DNA (ufDNA) of 33 breast cancer patients before and after ACHT. MCF-7 cells and PBMN from normal donors were exposed in vitro to melphalan, with or without amifostine. Results We observed at least one MSI event in PBMN cells, pfDNA or ufDNA of 87, 80 and 80% of patients, respectively. In vitro, melphalan induced MSI in both MCF-7 and normal PBMN cells. In PBMN cells, ACHT-induced MSI occurred together with a significant decrease in the expression of the DNA mismatch repair gene hMSH2. Amifostine decreased hMSH2 expression and also prevented MSI induction only in normal PBMN cells. Conclusions ACHT induced MSI in PBMN cells and in tumour-derived pfDNA. Because of its protective effect against ACHT induction of MSI in normal PBMN cells in vitro, amifostine may be a potential agent for preventing secondary leukaemias in patients exposed to ACHT.

Identification of a myeloid committed progenitor as the cancer-initiating cell in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by a block in differentiation and accumulation of promyelocytes in the bone marrow and blood. The majority of APL patients harbor the t(15: 17) translocation leading to expression of the fusion protein promyelocytic-retinoic acid receptor alpha. Treatment with retinoic acid leads to degradation of promyelocytic-retinoic acid receptor alpha protein and disappearance of leukemic cells; however, 30% of APL patients relapse after treatment. One potential mechanism for relapse is the persistence of cancer ""stem"" cells in hematopoietic organs after treatment. Using a novel sorting strategy we developed to isolate murine myeloid cells at distinct stages of differentiation, we identified a population of committed myeloid cells (CD34(+), c-kit(+), Fc gamma RIII/II(+), Gr1(int)) that accumulates in the spleen and bone marrow in a murine model of APL. We observed that these cells are capable of efficiently generating leukemia in recipient mice, demonstrating that this population represents the APL cancer-initiating cell. These cells down-regulate the transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) possibly through a methylation-dependent mechanism, indicating that C/EBP alpha deregulation contributes to transformation of APL cancer-initiating cells. Our findings provide further understanding of the biology of APL by demonstrating that a committed transformed progenitor can initiate and propagate the disease. (Blood. 2009; 114: 5415-5425)

Translocations t(X;14)(q28;q11) and t(Y;14)(q12;q11) in T-cell prolymphocytic leukemia

P>We report a case of T-cell prolymphocytic leukemia (T-PLL) in a 41-year-old male. Classical cytogenetic, spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) studies of a blood sample obtained at diagnosis revealed the co-existence of t(X;14)(q28;q11), t(Y;14)(q12;q11) and a ring chromosome derivated from i(8)(q10). Immunophenotypic studies revealed involvement of T-cell lineage, with proliferation of CD4(-) CD8(+). The co-existence of two translocations involving both sex chromosomes in a case of T-PLL is rare. Chromosomal instability associated with the disease progression may have allowed the emergence of cell clones with translocations involving the sex chromosomes and the ring chromosome observed.

High expression of AURKA and AURKB is associated with unfavorable cytogenetic abnormalities and high white blood cell count in patients with acute myeloid leukemia

In the present study, we analyzed AURKA and AURKB gene expression in 70 acute myeloid leukemia (AML) patients. There was no difference between leukemic samples and bone marrow mononuclear cells (BMMCs, n = 8) or CD34(+) progenitors (n = 10) from healthy donors. High white blood cells (WBC) counts were observed in the AURKA(+) and AURKB(+) groups, but no significant differences regarding age, gender, platelet counts or frequency of FLT3-ITD mutations. AURKA, but not AURKB, expression was independently associated with high WBC counts (OR: 3.15, 95% CI 1.07-9.24, p = 0.03). Moreover, the majority of cases that overexpressed AURKA and AURKB presented unfavorable cytogenetic abnormalities (p < 0.001). In conclusion, we described a significant association between overexpression of AURKA/B and cytogenetics findings in AML, which may be relevant to new therapeutic approaches, based on Aurora kinase inhibitors. (C) 2010 Elsevier Ltd. All rights reserved.

Association of drug metabolism gene polymorphisms with toxicities, graft-versus-host disease and survival after HLA-identical sibling hematopoietic stem cell transplantation for patients with leukemia

Individual differences in drug efficacy or toxicity can be influenced by genetic factors. We investigated whether polymorphisms of pharmacogenes that interfere with metabolism of drugs used in conditioning regimen and graft-versus-host disease (GvHD) prophylaxis could be associated with outcomes after HLA-identical hematopoietic stem cell transplantation (HSCT). Pharmacogenes and their polymorphisms were studied in 107 donors and patients with leukemia receiving HSCT. Candidate genes were: P450 cytochrome family (CYP2B6), glutathione-S-transferase family (GST), multidrug-resistance gene, methylenetetrahydrofolate reductase (MTHFR) and vitamin D receptor (VDR). The end points studied were oral mucositis (OM), hemorrhagic cystitis (HC), toxicity and venoocclusive disease of the liver (VOD), GvHD, transplantation-related mortality (TRM) and survival. Multivariate analyses, using death as a competing event, were performed adjusting for clinical factors. Among other clinical and genetic factors, polymorphisms of CYP2B6 genes that interfere with cyclophosphamide metabolism were associated with OM (recipient CYP2B6*4; P=0.0067), HC (recipient CYP2B6*2; P=0.03) and VOD (donor CYP2B6*6; P=0.03). Recipient MTHFR polymorphisms (C677T) were associated with acute GvHD (P=0.03), and recipient VDR TaqI with TRM and overall survival (P=0.006 and P=0.04, respectively). Genetic factors that interfere with drug metabolisms are associated with treatment-related toxicities, GvHD and survival after HLA-identical HSCT in patients with leukemia and should be investigated prospectively.

Detection of Clonal Immunoglobulin and T-Cell Receptor Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia Using a Low-Cost PCR Strategy

Background Imunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD) which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL) We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method Here we report on gene rearrangement frequencies and offer guidelines for the application of the technique Procedure Two hundred thirty three children had DNA extracted from bone marrow Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR PCR products were submitted to homo/heteroduplex analysis A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions Results At least one clonal marker could be detected in 98% of the patients and two markers in approximately 80% The most commonly rear ringed genes in precursor B cell ALL were IgH (75%) TCRD (59%) IgK (55%), and TCRG (54%) The most commonly rearranged genes for TALL were TCRG (100%) and TCRD (24%) The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells Conclusions We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases In addition these reactions ire suitable for MRD monitoring especially when aiming the selection of patients with high MRD levels (>= 10(-2)) at the end of induction therapy Such an approach would be very useful in centers with limited financial resources Pediatr Blood Cancer 2010 55 1278-1286 (C) 2010 Wiley Liss Inc