Mariastefania Antica

A simple method for RNA isolation from formalin-fixed and paraffin-embedded lymphatic tissues.

Gene activation that lies beneath lymphoid cell differentiation has been one of the most explored issues in immunology in the recent years. However, the analysis of this molecular event in lymphoproliferative diseases is often hampered by the lack of fresh material. Most tissues available for routine histological investigation are formalin fixed and paraffin embedded. Gene expression in such specimens could be analyzed using reverse transcription of mRNA and the polymerase chain reaction (RT-PCR). Therefore we adjusted and established a method for mRNA isolation from such specimens by a combination of previously reported protocols and a modification of the phenol/chloroform extraction method. Given the significance of transcription factors in the human hemopoietic system, we investigated whether mRNA could be successfully isolated from archival tissue for a study on expression of Ikaros family transcription factors in lymphatic tissue. Although quantitative analysis of RNA isolated from archival tissue is probably not feasible due to the unpredictable degree of RNA isolation varying from sample to sample, we show here that screening analysis is possible and simple.

Ikaros family transcription factors in chronic and acute leukemia

lkaros, Aiolos, and Helios are transcription factors important in lymphocyte differentiation and development [1-6]. One of the most interesting aspects of the Ikaros family members is that they are expressed as multiple splice variants and proteins arising from alternatively spliced transcripts may act as dominant negatives. Although expression of these short isoforms has been linked to lymphoid and myeloid leukemia [7-11] the comparison of Ikaros members mRNA expression level has not been reported. Here we show, by means of real time qRT-PCR method, a quantitative distribution of Ikaros, Aiolos, and Helios mRNA in hemopoietic cells of patients with lymphocytic leukemia, represented by the most frequent B cell chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (T cell ALL and common ALL), and compared with the acute leukemia of the myeloid lineage, acute myeloid leukemia (AML). We found that the relative quantity of Ikaros and Helios mRNA was similar in all subgroups. However, analysis of Aiolos showed a clear difference among groups because of its lower expression in all types of acute leukemia investigated. Our results associating a lower Aiolos expression with acute leukemia point to Aioloss role in human lymphocyte development, as previously shown in mice.

Functional Differences of T cells in B-Chronic Lymphocytic Leukemia

B cell chronic lymphocytic leukemia (B-CLL) is a disease characterized by an accumulation of monoclonal lymphocytes of B cell origin. Although the neoplastic process involves the B lymphocyte compartment, phenotypic and functional defects within the T lymphocyte population implicate their possible role in the pathogenesis of the disease. We analyzed the functional and morphological integrity of T lymphocytes from the peripheral blood of 64 patients with B-CLL. The activation of B-CLL T cells after PHA stimulation was determined by measuring [3H]-thymidine incorporation, assessing cell numbers in parallel cultures, and by monitoring the lymphocyte subsets during 9 days of cultivation. Our results indicate the presence of three functionally different populations of T cells in the peripheral blood of B-CLL patients. We present evidence for an increased proliferative potential of T lymphocytes from a group of patients with B-CLL.

Prolegomenon for Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is a unique lymphoproliferative disorder that scarcely occurs under the age of 40; thereafter the incidence of CLL increases exponentially with age. CLL is characterized by progressive expansion of malignant CD5+ME+ B‐cell clone accompanied by a myriad of cellular and humoral immune defects. Each of them might be linked to different clinically manifested complications such as increasing rate of infections, autoimmune disorders and disturbed immune surveillance against tumour cells. We assume that CLL occurs as a consequence of age‐dependent, genetically related functional restrictions of the thymic microenvironment in supporting common lymphoid progenitor cells (CD5+ME+CD4–CD8–) to differentiate into mature T‐cell and B‐cell descendants. In conjunction with genetic abnormalities developing in B‐cell progenitors, presumably expressing P glycoprotein (Pgp+), we postulate that developmentally altered T‐cell descendants, along with quantitative imbalance among CD4+, their subsets and CD8+ lymphocytes in the peripheral blood, play an important additional role in facilitating the malignant B‐cell clone emergence and in modulating the CLL clinical evolution. Namely, imbalance of any of T‐cell‐mediated cell interactive homeostatic mechanisms accompanied by imbalance in the production of various cytokines might in CLL influence leukaemic B‐cell growth by deregulating inducer (c‐myc and p53) and/or suppressor (bcl‐2 and mutant p53) oncogenes responsible for the promotion or suppression of B‐cell mitogenesis that may in turn further contribute to their impaired differentiation and/or differentiation arrest. In conclusion, CLL might be interpreted as a primary immunodeficiency syndrome developing in elderly population due to gradually evolving restriction of genetically controlled programs in the thymic microenvironment responsible for irregular maturation of common lymphoid progenitor cells that constitutively express CD5 antigen and ME receptor into T‐cell and B‐cell descendants.

Monoclonality in Helicobacter pylori-positive gastric biopsies: an early detection of mucosa-associated lymphoid tissue lymphoma.

Mucosa-associated lymphoid tissue (MALT) is not present in healthy gastric mucosa, but it can develop in sites of long-persisting inflammation and is connected with the development of MALT lymphoma. A monoclonal lymphocyte population is one of the characteristics of such lymphomas. In this study we analyzed gastric biopsies (formalin fixed and paraffin embedded or frozen) in 93 patients with dyspepsia accompanied by Helicobacter pylori infection. We applied PCR and single-cell immunocytochemistry to detect the clonality of the gastric B-cell population. Immunocytochemistry performed on 33 frozen biopsies showed two samples with monoclonal pattern. PCR analysis of immunoglobulin heavy-chain (IgH) gene rearrangements revealed two monoclonal populations out of 161 biopsies from 60 patients. We conclude that PCR analysis was the most sensitive method, which gave us insight into the nature of the earliest stage of MALT lymphoma in gastric biopsies.

Notch pathway connections in primary leukaemia samples of limited size

BackgroundThe Notch pathway combined with other signalling molecules acts specifically for the development of each blood cell type and differentiation stage. A causative role of Notch dysfunction in leukaemia development has been found in many studies so, initially only for T- acute lymphoblastic leukaemia (T-ALL) but more recently also for B cell and myeloid leukaemia. The aim of our study is to introduce a method for multiple direct analysis of the Notch pathway partners in a population of only 500 or fewer cells. The notion of this method consists in gaining insight into gene expression at the level of the malignant clone population. A small number of cells is a significant limitation when working on primary cells either when freshly isolated or when analysed after several days in cocultures.MethodsThe primers were designed to avoid genomic amplification through the selection of 3′ and 5′ primers that hybridise with different exons. Cell lines and primary cells were collected and multiplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) performed on a descending number of cells, ranging from 2,500 cells up to 50 cells per sample, for the Notch pathway genes and other transcription factors important for cell differentiation. ImageJ program, STATISTICA 13.1 software package and Student’s t-test were used for statistical evaluation. We checked protein expression by western blot.ResultsWe characterised the gene expression levels of Notch, Ikaros and Parp genes in leukaemia cell lines of B and T origin and in primary leukaemia samples of limited size. We further compared our results to the cDNA analysis obtained by total RNA isolation from a large number of cells as routinely performed in clinical laboratories, and finally tested the method described on primary cells from leukaemia patients.ConclusionsThis rapid multiple gene expression analysis of a small population of cells provides efficient cell classification determining malignant changes as an important additional information for clinical leukaemia diagnostics as well as for in vitro studies of primary cells.

Effect of Notch and PARP Pathways’ Inhibition in Leukemic Cells

Differentiation of blood cells is one of the most complex processes in the body. It is regulated by the action of transcription factors in time and space which creates a specific signaling network. In the hematopoietic signaling system, Notch is one of the main regulators of lymphocyte development. The aim of this study was to get insight into the regulation of Notch signalization and the influence of poly(ADP-ribose)polymerase (PARP) activity on this process in three leukemia cell lines obtained from B and T cells. PARP1 is an enzyme involved in posttranslational protein modification and chromatin structure changes. B and T leukemia cells were treated with Notch and PARP inhibitors, alone or in combination, for a prolonged period. The cells did not show cell proliferation arrest or apoptosis. Analysis of gene and protein expression set involved in Notch and PARP pathways revealed increase in JAGGED1 expression after PARP1 inhibition in B cell lines and changes in Ikaros family members in both B and T cell lines after γ-secretase inhibition. These data indicate that Notch and PARP inhibition, although not inducing differentiation in leukemia cells, induce changes in signaling circuits and chromatin modelling factors.

Notch affects the prodifferentiating effect of retinoic acid and PMA on leukemic cells.

Notch proteins determine cell fate decisions in the development of diverse tissues. Notch has been initially found in T‐ALL but its role has been also studied in myelopoiesis and myeloid leukemias. Studies in different model systems have led to a widespread controversy as to whether Notch promotes or blocks myeloid differentiation. In this work, we evaluated the influence of Notch activation on leukemic cell differentiation along the monocytic and myelocytic pathway induced by phorbol 12‐myristate 13‐acetate (PMA) or all‐trans retinoic acid (ATRA). We observed that differentiation of the human myeloblastic cell line HL‐60 can be retarded or blocked by Delta/Notch interaction. ATRA induces complete remission in patients with acute promyelocytic leukemia, but it cannot completely eliminate the leukemic clone and to be effective it should be combined with chemotherapy. Our findings suggest that Notch signaling may contribute to the incomplete elimination of the leukemic cells after PMA or ATRA treatment and the blockage of Notch pathway may be beneficial in the treatment of myeloid leukemia.

Ikaros family transcription factors expression in rat thymus: detection of impaired development.

The expression of Ikaros family transcription factors and consequently their signalling pathway is limiting for hematopoietic and lymphocyte development in mice and human. Due to their importance, these transcription factors are highly homologous between species. As an initial approach to examining the possible involvement of Ikaros transcription factors in pathogenesis of rat lymphoid development, we analyzed the expression of all known Ikaros family members, Ikaros, Aiolos, Helios, Eos and Pegasus in the rat thymus. We established a semi-quantitative RT-PCR to detect mRNA of each transcription factor. For the first time we give evidence of the expression of Ikaros family transcription factors in the rat thymus. Further, we evaluated whether their mRNA expression was succumbed to changes when the rats were exposed to ethanol, as a known debilitating agent during development. Therefore we analyzed the thymus of adult rats whose mothers were forced to drink ethanol during gestation, to detect possible changes in thymus mRNA expression levels of Ikaros, Aiolos, Helios, Eos and Pegasus. We found that rats prenatally exposed to ethanol show a slightly higher expression of Ikaros family transcription factors in the adult thymus when compared to control rats, but these differences were not statistically significant. We further studied the distribution of the major lymphocyte subpopulations in the rat thymus according to CD3, CD4 and CD8 expression by four color flow cytometry. We found a higher incidence of CD3 positive cells in the double positive, CD4+CD8+ thymic subpopulation of rats prenatally exposed to ethanol when compared to non-exposed animals. Our findings indicate that ethanol exposure of pregnant rats might influence the development of CD3 positive cells in the thymus of the offspring but this result should be further tackled at the level of transcription factor expression.