Biomedical subjects
Publications and source records attributed to G Emilia.
Human lymphocytes have been used by several researchers to investigate the biological effect of electromagnetic fields (EMF). EMF modulate the response by lymphocytes to lectin stimulation. The size and direction of the effect depends both on the lymphocyte physiology and on the physical parameters characterizing the EMF. Lymphocytes have also been used to investigate the genotoxicity of EMF exposure.
To date, the morphological aspects of sinus histiocytosis with massive lymphadenopathy (SHML) have been fully described. The disease is characterized by an enlargement of lymph nodes in which the sinuses are dilated and infiltrated by histiocytes, often phagocytosing lymphocytes. Even if the prognosis is usually benign and not requiring therapy, several fatal cases have been reported. The etiology is still obscure and the biology is not yet completely clear. Recent immunophenotypical studies suggest that histiocytes may belong to the T-zone associated histiocyte lineage. They may be cytologically homogeneous, but can express different antigenic patterns according to their stage of differentiation. Cytogenetic and molecular aspects of the disease have only been sporadically investigated. In order to better understand the pathogenesis of SHML, which seems to be a disorder lying in between the fields of infections, immunological disease and neoplasia, it is considered very useful to systematically employ a variety of immunophenotypical, cytogenetic and molecular techniques to study the disease, particularly in cases which are clinically atypical or with a more aggressive evolution.
C3H mice have been used to investigate the effect of a combination of cyclophosphamide (CY) and electromagnetic fields (PEMF). Mice were injected i.p. with a single dose of 200 mg/kg body weight of CY and then exposed to PEMF 24 h per day. In an initial series of experiments immediately after CY injection mice were exposed to PEMF until sacrifice. WBC counts in the peripheral blood demonstrated a quicker decline in WBC at days 1 and 2 in mice exposed to PEMF. Groups of mice were sacrificed at days 1, 4, 6, 8, and 10 after CY injection. In mice exposed to PEMF the spleen weight was less than in controls at days 6, 8, and 10. Autoradiographic studies demonstrated that the labeling index of bone marrow smears did not significantly differ between controls and experimental mice exposed to PEMF, whereas the spleen labeling index proved to be higher among control mice versus mice exposed to PEMF at day 6, and higher among mice exposed to PEMF versus controls at day 8. In a second series of experiments mice were exposed to PEMF only over the 24 h following CY injection. We found that the spleens of mice exposed to PEMF weighed less than those of controls at days 6 and 8. The labeling index of bone marrow did evidence a slight decrease among mice exposed to PEMF at days 8 and 10 after CY injection versus control mice. The spleen labeling index proved to be lower in experimental mice exposed to PEMF than in controls at days 4, 6, and 8. Mice were then injected with CY, half were exposed to PEMF, and 24 h later bone marrow was recovered from both groups of animals. The same number of bone marrow cells was injected via the tail vein into recipient mice irradiated to 8.5 Gy. The grafting efficiency of the bone marrow was evaluated by examining the number of spleen colonies and the spleen and bone marrow labeling indices at day 8; all parameters proved to be significantly lower among mice grafted with the bone marrow of mice injected with CY and exposed to PEMF than among controls injected with CY only. Finally, we found th at the effect of PEMF is evident only if mice are exposed during the 24 h following CY injection. The data reported here indicates that PEMF exposure after CY injection increases the damage induced in mice by CY.
We describe a case of "sinus histiocytosis with massive lymphadenopathy" (SHML) studied by immunohistochemical, cytogenetic and molecular analysis. The immunophenotyping showed that the lymph node histiocytes were strongly positive for the S-100 protein and MoAb LeuM3, OKM5, KP1 and DRC-1; a portion of these cells was also positive for OKT6 and Leu3A, suggesting a possible relationship with the veiled cells, which represent an intermediate step in the pathway from the Langerhans cell to the interdigitating reticulum cell. Cytogenetic analysis showed a normal prevalent clone and a small hypodiploid clone and the molecular study showed no detectable involvement of the c-fms proto-oncogene, which is related to monocyte/macrophages. Unfortunately all these data do not seem sufficient to define the benign or neoplastic nature of the disease. Further investigations, immunophenotypical, cytogenetic and molecular, are needed to elucidate the pathogenesis of the disease, especially for more aggressive cases or for cases with unfavorable evolution.
Northern blot analysis of four typical cases of acute promyelocytic leukemia showed that one of the cell population examined was characterized by a very high level of expression of the myeloperoxidase (MPO) gene. Western blot analysis confirms that the protein content of the cells corresponded to the levels of the MPO mRNA. Southern blot studies of the DNA of this cell population ruled out the presence of any genome amplification or rearrangement. Chromosome hybridization studies in situ confirmed that the MPO gene was translocated on the long arm of chromosome 15. The observation that a typical genomic pattern may or may not be associated with the MPO overexpression leads us to believe that so far it is impossible to reach any conclusion about the significance of the translocation in the genesis of MPO overexpression.
Two patients with Ph1-positive chronic myelogenous leukemia with pleural blastic transformation occurring before medullary involvement are presented. The clonal origin of the pleural cells identified as unclassified blasts in 1 patient and as erythroid blasts in the other was confirmed by the presence of the t(9;22) translocation and their clonal evolution by the presence of duplicated Ph1 and additional chromosome alterations. DNA obtained from the pleural blasts and peripheral blood cells of 1 patient showed an identically rearranged bcr configuration, indicating the origin of the pleural blasts from the CML clone and suggesting that this genomic event is not directly linked with the progression of disease.
The hybridization to a complementary RNA (cRNA) probe both in situ and in solution was used to assay tiny amounts of mRNA of the lactoferrin (LF) and myeloperoxidase (MPO) genes in normal bone marrow cells and in acute and chronic lymphoid leukemias. Evidence is reported that this technique is much more sensitive than the standard Northern blot technique. The LF mRNA was detectable in three of seven cases of acute lymphoblastic leukemia (ALL) and in three of seven cases of chronic lymphocytic leukemia (CLL). Four cases of ALL were also positive when tested with the MPO cRNA. It is apparent from these results that myeloid specific mRNA, different from MPO, may be detected in leukemic cells with lymphoid phenotype using a method more sensitive than the Northern blot technique. Whether or not the molecular events observed in these cell populations reflect events physiologically occurring rather than a deregulation of gene expression associated to leukemogenesis remains to be established.
The Philadelphia (Ph1) chromosome is found in the majority of patients affected by chronic myelogenous leukemia (CML), being considered the hallmark of the disease, but around 5-8% of patients diagnosed as CML lack the Ph1 chromosome-negative (Ph-) CML has been discussed extensively in the literature because of its heterogeneity. However, it is now accepted that some of the Ph1-CML patients have a disease indistinguishable from Ph1-positive (Ph+) CML. It was investigated whether Ph- CML with clinical and morphological features indicating true CML would always have bcr rearrangements, as the relocation of c-abl from 9q34 into the breakpoint cluster region on 22q11 is considered a crucial event in the pathogenesis of CML. From molecular studies, it seemed that Ph- CML with features of true CML always have the bcr rearrangement, while Ph- patients, lacking such rearrangement, have atypical forms of CML. Here we describe 8 Ph- CML and myeloproliferative syndrome (MPS) patients of whom 6 were by all respects true CML cases. Nevertheless, bcr rearrangement and expression of the classic bcr/abl chimeric mRNA was found in only 1 of the 6 patients. More advanced molecular techniques will be needed to understand which molecular mechanisms underlie Ph-, bcr- CML, resulting in phenotypes sometimes indistinguishable from Ph+, bcr+ CML.
Cytogenetic and molecular data of three patients affected by primary myelofibrosis with myeloid metaplasia (PMMM) evolving to blastic crisis are reported. The cytogenetic findings were uncommon. The first patient (female) showed an idic(X)(q13) as the sole alteration in chronic phase, with an additional r(7) in 67% of the cells of the blast crisis; the other two patients showed, in blast crisis, a partial trisomy of the long arm of chromosome 1, without translocation, as a unique structural abnormality. These findings confirm the presence of nonrandom, although nonspecific, alterations in PMMM that, in our cases, seem to be related to the multistep progression of the neoplastic process. Molecular investigations have been applied to study the genomic organization and the level of expression of genes such as bcr and calcyclin and c-fms protooncogene possibly involved in the molecular mechanisms underlying cell proliferation in hematopoietic cells. The data obtained are discussed with respect to the myeloproliferative disorder.
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Two further cases of acquired isodicentric (X)(q13) chromosome in patients with myeloproliferative disorders are reported. One patient had a primary myelofibrosis and the other a chronic myelogenous leukemia. DNA replication study demonstrated that both abnormal X chromosomes were late replicating in all cells examined. The inactive condition of idic(X)(q13) seems to be indicative of a selective advantage for a cell carrying this alteration.
The expression of the myeloperoxidase (MPO) gene was studied, by means of Northern blot analysis in 14 cases of acute myeloid leukemia (AML), 11 cases of chronic myeloid leukemia (CML), and 6 cases of CML blast crisis, and in HL60 cells before and after induction of terminal differentiation with retinoic acid (RA), phorbol esters (TPA), or vitamin D. The expression of a panel of cell cycle-related genes, namely C-MYC, histone H3, ornithine decarboxylase, P53, vimentin, and calcyclin, was also studied in the same cell populations. Our results indicate that: (a) MPO gene expression (steady state mRNA levels) is strictly confined to the first stages of myeloid differentiation, reaching its peak at the promyelocyte stage and becoming undetectable in mature granulocytes and monocytes; (b) cells devoid of any detectable MPO enzymatic activity such as leukemic basophils have a high content of MPO mRNA; and (c) MPO gene expression is not related to the growth activity of the cell population. Finally, our results show that the pattern of expression of growth-regulated genes in the neoplastic myeloid disorders AML, CML, and CML blast crisis is remarkably different.
C3H/Km flora-defined mice were used to investigate the effect of exposure to pulsing electromagnetic field (PEMF) after total body x-ray irradiation. Prolonged exposure to PEMF had no effect on normal nonirradiated mice. When mice irradiated with different doses of x-ray (8.5 Gy, 6.8 Gy, and 6.3 Gy) were exposed to PEMF 24 h a day, we observed a more rapid decline in white blood cells (WBC) in the peripheral blood of mice exposed to PEMF at all the x-ray dosages used. No effect of exposure to PEMF was observed on the survival of the mice irradiated with 6.3 Gy and 8.5 Gy; in mice irradiated with 6.8 Gy, 2 out of 12 survived when exposed to PEMF as compared to 10 out of 12 control mice that were irradiated only. At day 4 after irradiation autoradiographic studies performed on bone marrow and spleen of 8.5-Gy-irradiated mice showed no difference between controls and mice exposed to PEMF, whereas on 6.8-Gy mice the bone marrow labeling index was lower in mice exposed to PEMF. In mice irradiated to 6.3 Gy we observed that the recovery of WBC in the peripheral blood was slowed in mice exposed to PEMF and their body weight was significantly lower than in control mice that were irradiated only. The spleen and bone marrow of the mice irradiated to 6.3 Gy and sacrificed at days 4, 14, 20, and 25 after irradiation were analyzed by autoradiography to evaluate the labeling index. Half of the spleens from mice sacrificed at day 25 after irradiation were used to evaluate the RNA content. Autoradiography showed that in the spleen and bone marrow of control mice, there were more cells labeled with [3H]thymidine at days 4 and 14 and less at days 20 and 25 after irradiation in comparison with mice irradiated and exposed to PEMF. The Northern blot analysis of histone H3 and p53 protein RNAs extracted from the spleens at day 25 after irradiation showed a slight increase in cycling cells among spleens of mice exposed to PEMF. We suggest that the exposure to PEMF immediately after x-ray irradiation results in increased damage.
Despite the major breakthrough in the knowledge of the molecular events underlying the t(9;22) translocation, still no consistent data have been found on the evolution of Ph1 positive CML from the chronic to the accelerated or blastic phase of the disease. In most patients in fact the bcr/abl rearrangements are identical both in chronic phase and in blast crisis, and overall differences in chronic phase duration, related to different location of breakpoints inside the bcr region, were found to be marginal. We approached this problem by studying the molecular features of the bcr/abl abnormality in rare CML patients with very long, atypical chronic phase. The three patients studied, whose chronic phase duration is 17, 19, and 21 years, respectively, have typical genomic bcr rearrangements, and two of them show, hybridizing Northern blots to c-abl, the 8.5 kb mRNA, as that typically present in CML. It seems that genomic alterations within bcr and abl cannot account, alone, for the duration of the chronic phase of Ph1 positive CML and those quantitative and/or qualitative alterations of the p210 bcr/abl protein, unluckily awkward to prove, might be responsible for the atypical clinical features of these CML long survivors.
We have established a new human plasma cell line from the peripheral blood of a patient with an IgA-kappa plasma-cell leukemia. Morphological, immunological, cytogenetic and molecular studies confirm that the cultured cells are derived from the same clone of leukemic plasma cell in vivo. The established cell line (MT3) grows in suspension, secretes high amounts of IgA kappa and exhibits morphological and ultrastructural characteristics of plasma cells. Surface marker analysis shows that both primary and cultured cells express the plasma-cell-associated antigens PCA-1 and T10, while specific B- and T-cell determinants and EBV nuclear antigen are undetectable. In the established cell line a few cells express Ia-like and CALLA antigens. Cytogenetic analysis of MT3 cells reveals a prevalent hypertriploid karyotype with constant chromosomal aberrations consisting of 14q+, 22q- and marker chromosomes.
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A B-cell chronic lymphocytic leukaemia terminated, 5 years from the onset, with a blast crisis. Karyotype analysis showed that the terminal lymphoblastic population evolved from the original B lymphocytic clone. The levels of expression of several oncogenes, as well as the mu chain gene, were assayed by Northern blot hybridization analysis of RNA extracted from the lymphoid populations before and after the 'acute transformation'. A seven- to eight-fold increase in the expression of c-myc and mu chain genes was observed in the blast population. c-myb, c-fes, c-Haras were not expressed either before or after the blastic transformation.
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