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D Loukopoulos

Publications and source records attributed to D Loukopoulos.

At least 55 records · Page 3Linked to original sources
PubMed1997

A novel chromosomal abnormality involving chromosomes 2 and 18 in a patient with myelodysplastic syndrome.

Cytogenetic analysis of bone marrow cells from a patient with myelodysplastic syndrome associated with eosinophilia showed a complex translocation with a 46,XY,t(2;18;2)(p23;q11;q32) karyotype. The patient has refractory anemia (RA) according to the French-American-British Cooperative Group (FAB) classification, and after 90 months of follow-up he shows no evidence of leukemic transformation. This chromosomal abnormality has not been previously described in myelodysplastic syndromes and may be associated with good prognosis as the patient has been stable for a long time.

Adult↗
PubMed1997

t(14;18) chromosomal translocation in follicular lymphoma: an event occurring with almost equal frequency both at the D to J(H) and at later stages in the rearrangement process of the immunoglobulin heavy chain gene locus.

bcl-2/IgH fusion is considered a genetic error which occurs at the diversity (D) to joining (J(H)) stage of the gene rearrangement process in the immunoglobulin heavy chain (IgH) gene locus. Translocations of the bcl-2 protooncogene to the IgH locus at ontogenetically later IgH gene rearrangements are thought to represent exceptions. In the present study we analysed the junctional nucleotide sequence of 18 bcl-2/IgH fusion genes identifiable by polymerase chain reaction performed on DNA extracted from diagnostic lymph node tissue of 14 follicular lymphoma patients. In all clones studied, segments of variable length were found interposed between bcl-2 and J(H) gene sequences. Nucleotide sequence data analysis and comparisons performed with the corresponding germline sequences using the GenBank/EMBL database revealed the presence of D segments in most of the bcl-2/IgH fusion genes under study (13/18). By the same kind of computer-aided analysis, previously unrecognized D segments were identified in many published junctional sequences. These results suggest that bcl-2/IgH fusion events are very prevalent in rather more differentiated stages in B-cell ontogeny than previously recognized.

Base Sequence↗
PubMed1997

Follicular lymphoma immunoglobulin kappa light chains are affected by the antigen selection process, but to a lesser degree than their partner heavy chains.

Follicular lymphoma cells carry surface immunoglobulin whose heavy chain variable (VH) regions exhibit considerable divergence from the aminoacid sequence predicted by the germline nucleotide sequence as a result of the somatic hypermutation process. The present study examined the extent of somatic hypermutation in follicular lymphoma kappa light chain variable region (V kappa) genes about which the available data is limited. DNA extracted from fresh frozen lymph node tissue of 14 patients with follicular lymphoma at diagnosis was subjected to polymerase chain reaction (PCR) amplification aimed at detecting clonal VH and VL (L: light chain) gene rearrangements. Clonal V kappa gene rearrangements were detected in 10/14 cases. Amplified VH and V kappa genes of these 10 cases were directly sequenced by the dideoxy-chain termination method. In all cases, rearranged VH genes demonstrated numerous mutations clustering in the complementarity determining regions (CDRs), in keeping with previous reports. The degree of divergence of the rearranged V kappa genes from the closest homologous germline V kappa genes varied significantly. Furthermore, two patterns of mutations were observed: (i) in six cases (60%), mutations were most often of the replacement (R) type (changing the aminoacid sequence of the encoded polypeptide) in the CDRs and of the silent (S) type (leaving the aminoacid sequence of the encoded polypeptide unchanged) in the framework regions (FWRs) resulting in R:S ratios significantly greater than would have occurred by chance: (ii) in four cases (40%), very few or no mutations were observed and the distribution of mutations as well as the R:S mutation ratios did not differ significantly from what would have occurred by chance alone. These findings imply that, compared to their partner heavy chains, the kappa light chains of follicular lymphoma neoplastic B-cells' surface immunoglobulin (sIg): (i) are less affected by somatic hypermutation: (ii) play a less significant role in the antigen selection process.

Amino Acid Sequence↗
PubMed1997

Trisomy 8 in a patient who responded to therapy with all-trans-retinoic acid and developed paroxysmal nocturnal haemoglobinuria.

Trisomy 8 is the most common numerical chromosomal abnormality in myelodysplastic syndromes (MDS). Paroxysmal nocturnal haemoglobinuria (PNH) is an aquired haemolytic anaemia, clonal in nature, due to somatic mutation. PNH may evolve to aplastic anaemia, to MDS or to acute myeloid leukaemia. We present a patient who had trisomy 8 mosaicism at disease presentation who received therapy with all-trans-retinoic acid, responded to therapy, and developed PNH in the course of the disease. Cytogenetics at the time of PNH diagnosis showed a normal karyotype.

Adolescent↗
PubMed1997

Erythropoietin levels and microcytosis in heterozygous beta-thalassaemia.

Erythropoietin levels were determined in 50 Greek females: 20 beta-thalassaemia (beta-thal) heterozygotes, 15 with a diagnosis of iron-deficiency anaemia and 15 normal controls. In beta-thal trait carriers, the erythropoietin levels were slightly higher than in normal controls (16.65 +/- 4.43 vs. 12.84 +/- 2.47 mU/ml); these levels were significantly lower than those in iron-deficient subjects with the same degree of anaemia (55.24 +/- 31.35 mU/ml). In both groups, the erythropoietin levels are statistically correlated with the severity of anaemia (r = -0.537 p < 0.05 for iron deficiency; r = -0.610 p < 0.01 for beta-thal heterozygotes). In beta-thal heterozygotes, a close inverse correlation with red cell number and erythropoietin levels was also noted. It is suggested that microcytosis accompanying beta-thal trait constitutes an additional factor intervening in the regulation of erythropoiesis.

Adult↗
PubMed1997

Detection of "PNH red cell" populations in hematological disorders using the Sephacryl Gel Test micro typing system.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder characterised by an unusual sensitivity of abnormal red cell population(s) to complement lysis, due to a complete or incomplete defect of various surface molecules, including CD55 and CD59. PNH has been associated with various hematological disorders. Using a newly introduced method, the Sephacryl gel test microtyping system, we investigated the presence of CD55 or CD59 defective red cell populations in several hematological disorders. It was also found that a large proportion of such patients possess CD55 deficient populations, while a smaller but still significant proportion possess CD59 deficient populations. Defective red cell populations were detected in normal subjects as well. These findings need further investigation. Nevertheless the Sephacryl Gel Test microtyping system although non specific, seems to be useful in screening for the PNH and/or "PNH-like" red cell defect in several hematological disorders.

CD55 Antigens↗
PubMed1997

Modulation of globin gene expression in cultured erythroid precursors derived from normal individuals: transcriptional and posttranscriptional regulation by hemin.

We are interested in the genetic mechanisms whereby several classes of drugs increase fetal hemoglobin (HbF) in patients with sickle-cell anemia or beta-thalassemia. Recently, we have shown (Kollia et al., Proc. Natl. Acad. Sci. U.S.A. 93: 5693, 1996) that cultured primary human adult erythroid cells (hAEC) offer a useful model for the study of transcriptional and posttranscriptional regulation of globin gene expression. We have found also that hemin markedly increases HbF levels in these cells. We report here the effect of hemin on globin gene transcription and RNA processing in hAEC. Quantitative reverse transcriptase-polymerase chain reaction analysis showed that the gamma-globin message levels in the cytoplasm and nucleus were increased two-fold by hemin. In the untreated cells, only spliced gamma-transcripts were detected in the cytoplasm, indicating that only completely processed gamma-RNA is transported to the cytoplasm, whereas approximately half of the nuclear gamma-globin RNA transcripts were unspliced. After treatment with hemin, correctly spliced gamma-transcripts increased in the cytoplasm and nucleus, while the unprocessed gamma-transcripts decreased in number in the nucleus. We also studied epsilon-globin RNA transcripts; in the cytoplasm of untreated cells, only correctly processed transcripts were present, whereas the nuclear epsilon-globin RNA transcripts were unspliced. In hemin-induced cells, unspliced nuclear epsilon-transcripts decreased in number. In contrast to the gamma- and epsilon-globin genes, the levels of full-length, correctly spliced beta-globin message are not affected by hemin. Nuclear run-on transcription assays confirmed the increase in the rate of transcription of gamma- and epsilon-globin genes in hemin-treated versus untreated hAEC. These results indicate that hemin affects the expression of embryonic and fetal globin genes by acting both at the transcriptional and posttranscriptional levels. These results may be relevant to the action of other agents that affect the hemoglobin phenotype of human erythroid cells.

Adult↗
PubMed1997

New therapies for the haemoglobinopathies.

Re-activation of the fetal globin genes is the most realistic approach to correct the deranged pathophysiology of the haemoglobinopathies because the presence of gamma-chains can neutralize the toxic effects of the unbound alpha-globin chains in the beta-thalassaemias and inhibit the polymerization of Hb S in the sickle cell syndromes. Re-induction of fetal haemoglobin synthesis can be brought about either by direct activation of the respective promoter genes and possibly other positively acting elements, or by recruitment into proliferation and differentiation of a population of erythroid precursors which retain the gamma-chain synthesis programme but remain dormant in the bone marrow of the adult unless called up in cases of acute erythroid expansion. Examples of the first group include the butyric acid and derivatives and 5' azacytidine. The second group comprises erythropoietin and a series of cytostatics, with hydroxyurea as the main representative. The activity of most of the above agents has already been studied in cell cultures and animals and confirmed in several patients, both at the haematological and biochemical level as well as through their frank clinical improvement. However, application of these drugs at large is not yet justified because a series of questions concerning their long-term efficacy, the correct dosage and timing, their tolerance and toxicity, and the potential long-term dangers, including mutagenicity are still unresolved.

Anemia, Sickle Cell↗
PubMed1996

Molecular demonstration of BCR/ABL fusion in two cases with chronic myeloproliferative disorder carrying variant Philadelphia t(14;22)(q32;q11).

We report two cases with chronic myeloproliferative disorder which were found to carry simple variant Philadelphia (Ph) t(14;22)(q32;q11) in unstimulated bone marrow mononuclear cells. Both cases were characterized molecularly by Southern blot, reverse transcription-polymerase chain reaction (RT-PCR), and direct sequencing of the RT-PCR products. In the first case (female, aged 65, in blastic transformation which developed one year after the initial diagnosis of myelofibrosis), a t(14;22) (q32;q11) was found in association with several other chromosomal abnormalities [48,XX,+X,+5,del(5) (q12q32),+8,der(9)t(9;11)(q32;q11),-11]; molecular analysis demonstrated the presence of a BCR-ABL chimeric gene and mRNA transcript of the b2-a2 type. In the second case (female, aged 16, with clinical and hematologic features typical of chronic myelogenous leukemia in chronic phase), a t(14;22) (q32;q11) was identified as the sole karyotypic abnormality; again, molecular analysis demonstrated the presence of a BCR-ABL chimeric gene and mRNA transcript, this time of the b3-a2 type. Our findings further support the notion that, even when undetectable by conventional cytogenetics, band 9q34 participates in all Ph chromosomes and leads to the formation of chimeric BCR-ABL genes.

Adolescent↗
PubMed1996

Development of plasma cell tumors during treatment of multiple myeloma.

Plasma cell tumors (plasmacytomas-PCT) of the bone, or extramedullary PCT, may be diagnosed in patients with or without the diagnostic criteria for systemic multiple myeloma (MM). The reason for the local development of these tumors is not clear. Recent reports emphasize the contribution of CT and MRI in the detection of bone lesions and their expansion into the soft tissues. We report the development of PCT in nine patients with MM under maintenance treatment with alpha-IFN, of whom six had no evidence of systemic relapse and three had indications of early relapse. The PCT were located in the pelvis (4), thoracic (3), cervical (1), and lumbar (2) spine and in 8/9 cases were not demonstrable on plain X-rays. These observations suggest that frequent screening with advanced imaging techniques may detect local disease expansion in asymptomatic patients. Early application of radiochemotherapy may improve prognosis.

Adult↗
PubMed1996

Analysis of the kappa light chain variable region in multiple myeloma.

The study of immunoglobulin heavy chain gene rearrangements in multiple myeloma has revealed extensive divergence from the germline sequences, but no intraclonal diversity with disease evolution. Our study investigated the state of the rearranged kappa light chain variable region (V kappa) gene segments as well as abortive V kappa family gene usage in cases of multiple myeloma expressing lambda light chain. We studied 11 cases of kappa and five cases of lambda light chain-expressing multiple myeloma. Total cellular RNA was extracted from the bone marrow of patients with overt disease and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to amplify clonally rearranged variable region sequences. Direct nucleotide sequencing by the dideoxy-chain termination method was performed on the RT-PCR products. We did not observe preferential usage of certain V kappa gene families. Mutation frequencies of the V kappa segments varied in number. In the majority of cases, extensive somatic mutations occurred within the complementarity determining regions (CDRs) of V kappa, whereas only a limited degree of divergence from the germline was observed in others. In all cases studied. replacement mutations tended to cluster in the CDRs, a finding compatible with an antigen-driven somatic hypermutation process. In 3/5 cases of lambda light-chain expressing multiple myeloma, abortively rearranged V kappa gene segments were amplified from genomic DNA; in two cases a non-templated nucleotide insertion rendering the V kappa sequences out-of-frame was observed, and in the third a stop codon was identified in the open reading frame of the V kappa sequence. Somatic mutations were observed in all cases of abortive V kappa genes studied; however, their distribution does not suggest selection by antigen. We conclude that somatic mutations observed in the V kappa regions of myeloma cells are of variable extent and suggest operation of the antigen selection process. Lack of or minimal somatic hypermutation in a few cases may be in some way implicated in the biological heterogeneity of the disease.

Amino Acid Sequence↗
PubMed1996

The effect of GM-CSF on reticulocytes, haemoglobin and haematocrit in patients receiving chemotherapy for solid tumours.

The effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on reticulocytes, haemoglobin and haematocrit levels of patients receiving chemotherapy for solid tumours was assessed. Forty-two patients were randomized into two groups who either did or did not receive GM-CSF 300 U/d on days 8 and 15 post-chemotherapy for neutropenia prophylaxis. Patients were stratified according to tumour type (non small-cell lung cancer, small-cell lung cancer, ovarian and breast cancer). Eight patients with progressive disease were excluded. The 34 patients evaluated completed six chemotherapeutic cycles of 21 days. They did not receive transfusions or erythropoietin. The estimated dose intensities (mg/m2/d) for each drug used were significantly higher in the group on GM-CSF compared to the group without. Haemoglobin and haematocrit were measured by the H * 1 (Bayer) haematology analyser and reticulocyte counts by the flow-cytometric reticulocyte analyser, R-1000 (Sysmex). Measurements were conducted on days 1 and 12 of every cycle for six cycles. At the beginning of therapy there were no significant differences between the two groups. However, red cell and reticulocyte counts fell significantly in the group without GM-CSF compared to the group on GM-CSF. Reticulocyte differences preceded those of haemoglobin and haematocrit. The data indicate a positive in vivo effect of GM-CSF on the erythroid tissue of patients receiving chemotherapy for solid tumours.

Adult↗
PubMed1996

Evidence for immunoglobulin heavy chain variable region gene replacement in a patient with B cell chronic lymphocytic leukemia.

Immunoglobulin heavy chain variable (V) gene replacement is an unusual recombinatorial event characterized by rearrangement of a germline V gene to a preformed VDJ gene complex. This phenomenon has occasionally been implicated in the emergence of clonal subpopulations during the course of acute lymphoblastic leukemia; it has also been found in murine precursor B cell lines. V gene replacement has never been described in lymphoproliferative disorders corresponding to more differentiated stages of B cell ontogeny. The present communication provides evidence for the operation of the same mechanism in B cell chronic lymphocytic leukemia (B-CLL). Genomic DNA and total cellular RNA extracted from peripheral blood mononuclear cells of a 48-year-old female patient diagnosed as having typical B-CLL were subjected to polymerase chain reaction (PCR) amplification aiming to detect rearranged clonal heavy and light chain variable genes (VH and VL, respectively). PCR consistently gave two VH amplification products, both at the DNA and the RNA level; similar analysis for the VL region revealed the presence of a single rearranged VK gene. Direct sequence analysis of the PCR products revealed that, except for a number of silent mutations, the single rearranged VK gene was identical to the germline A1-A17 VK gene. The two rearranged VH gene segments belong to the VHl and VHIII gene families and are closely homologous, respectively, to the germline gene segments V1-18 and V3-30, which have been shown to be used by autoantibodies. Both rearranged VH genes showed identical in-frame D-N-JH junctions and JH gene usage (JH5b), whereas the VH-N-D junctions were different. The above findings indicate that, during the course of the disease of our patient, VH gene replacement took place giving rise to two different clonally related subpopulations. This raises the intriguing possibility that the recombinase machinery, which governs Ig recombinatorial processes, might be operative even at more advanced stages in B cell ontogeny.

Amino Acid Sequence↗