Estimates of Environmental Exposure to Radiofrequency Electromagnetic Fields and Risk of Lymphoma Subtypes.

We investigated the association between environmental exposure to radiofrequency electromagnetic fields (RF-EMF) and risk of lymphoma subtypes in a case-control study comprised of 322 patients and 444 individuals serving as controls in Sardinia, Italy in 1998-2004. Questionnaire information included the self-reported distance of the three longest held residential addresses from fixed radio-television transmitters and mobile phone base stations. We georeferenced the residential addresses of all study subjects and obtained the spatial coordinates of mobile phone base stations. For each address within a 500-meter radius from a mobile phone base station, we estimated the RF-EMF intensity using predictions from spatial models, and we performed RF-EMF measurements at the door in the subset of the longest held addresses within a 250-meter radius. We calculated risk of lymphoma and its major subtypes associated with the RF-EMF exposure metrics with unconditional logistic regression, adjusting by age, gender and years of education. In the analysis of self-reported data, risk associated with residence in proximity (within 50 meters) to fixed radio-television transmitters was likewise elevated for lymphoma overall [odds ratio = 2.7, 95% confidence interval = 1.5-4.6], and for the major lymphoma subtypes. With reference to mobile phone base stations, we did not observe an association with either the self-reported, or the geocoded distance from mobile phone base stations. RF-EMF measurements did not vary by case-control status. By comparing the self-reports to the geocoded data, we discovered that the cases tended to underestimate the distance from mobile phone base stations differentially from the controls ( P = 0.073). The interpretation of our findings is compromised by the limited study size, particularly in the analysis of the individual lymphoma subtypes, and the unavailability of the spatial coordinates of radio-television transmitters. Nonetheless, our results do not support the hypothesis of a link between environmental exposure to RF-EMF from mobile phone base stations and risk of lymphoma subtypes.

Plasma polychlorobiphenyl and organochlorine pesticide level and risk of major lymphoma subtypes.

BACKGROUND: There is conflicting epidemiological evidence concerning an increase in risk of non-Hodgkin's lymphoma (NHL) associated with elevated blood levels of persistent organochlorine (OC) pesticides and polychlorobiphenyls (PCBs). METHODS: We measured the concentration of 17 OC pesticides, including hexachlorobenzene (HCB), four lindane isomers (alpha-, beta-, gamma- and delta-hexachlorocyclohexane (HCH)), two chlordane species (heptachlor and oxy-chlordane), four cyclodiene insecticides (aldrin, dieldrin, endrin and mirex), six dichloro-diphenyl-trichloroethane (DDT) isomers and nine PCB congeners (PCBs 28, 52, 101, 118, 138, 153, 170, 180 and 194) in plasma samples of 377 subjects, including 174 NHL cases and 203 controls from France, Germany and Spain. The risk of NHL and its major subtypes associated with increasing blood levels of OC pesticides and PCBs was calculated using unconditional logistic regression. RESULTS: Risk of NHL, diffuse large B cell lymphoma (DLBCL) and chronic lymphatic leukaemia (CLL) did not increase with plasma levels of HCB, beta-HCH, p,p'-dichloro-diphenyl-dichloroethylene (DDE), or total and individual PCBs or their functional groups, in the overall study population. Substantial heterogeneity in DLBCL risk associated with immunotoxic PCBs (p = 0.03) existed between the Spanish subgroup (odds ratio (OR) for immunotoxic PCB plasma level above the median vs below the median was 0.7, 95% CI 0.3 to 1.6) and the French and German subgroups combined (OR 3.2, 95% CI 0.9 to 11.5). CONCLUSION: We did not find evidence of an association between NHL risk and plasma level of OC pesticides and PCBs.

The MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.

Tumor vascularity and tryptase-positive mast cells correlate with a poor prognosis in melanoma.

BACKGROUND: The importance of angiogenesis in melanoma has been controversial and is not homogeneous. Mast cell density (MCD) is highly correlated with the extent of both normal and pathological angiogenesis, such as that in chronic inflammatory diseases and tumours. METHODS: We evaluated the prognostic significance of tumour microvascular density (MVD) and MCD in 25 advanced melanoma patients after resection and a 4-5-year follow up: 48% of the patients were alive and free of metastases (good prognostic subgroup); 16% had developed regional nodal metastases (intermediate prognostic subgroup); and 36% had died (poor prognostic subgroup). Tissues samples were investigated immunohistochemically to count microvessels and mast cells with an antifactor VIII and an antitryptase antibody, respectively. RESULTS: Immunohistological staining showed a higher number of microvessels and mast cells in melanoma lesions of poor prognosis as compared with intermediate prognosis and with good prognosis, respectively. CONCLUSIONS: These data agree with those showing a close relationship between MCD and angiogenesis during tumour progression and demonstrate, for the first time, a prognostic significance of MCD in human melanoma.

Nonrandom chromosomal imbalances in primary mediastinal B-cell lymphoma detected by arbitrarily primed PCR fingerprinting.

We used arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting to identify chromosomal imbalances in six primary mediastinal B-cell lymphomas (PMBLs). Seventy-four chromosomal imbalances were detected, consisting of 49 sequence gains and 25 losses. Amplifications on chromosome X were seen in five cases, four of which involved the same chromosomal locus. Nonrandom gains at the same locus were also identified on chromosomes 2 and 7 in four cases and on chromosomes 5, 9, and 12 in three cases. Five PMBLs were also analyzed by comparative genomic hybridization (CGH), which found chromosome arm 9p amplification as the only nonrandom imbalance. Our data demonstrate that chromosomal amplifications outnumber losses in PMBL. These mainly involve chromosomes 9 and X and may reflect more complex phenomena, such as translocations or other chromosomal rearrangements, as AP-PCR found coexistent gains and losses on these chromosomes. Comparison between AP-PCR and CGH suggests that anomalies affecting the same chromosomal regions may occur at much higher frequencies than expected by CGH, suggesting that genomic amplifications are usually confined to DNA segments smaller than the megabase long segments required for detection in CGH. Modest increases in genetic material may be as effective as higher-level amplifications when affecting sites where a proto-oncogene resides.

Developmentally regulated expression and localization of fibroblast growth factor receptors in the human muscle.

Fibroblast growth factors (FGFs) are believed to play a key role in tissue differentiation and maturation. Thus, the expression of the four members of the high-affinity tyrosine kinase FGF receptor family (FGFRs) and of the low-affinity heparan sulphate proteoglycan binding sites, syndecan-1 and perlecan, was studied in the human skeletal muscle during development. Northern blot analysis demonstrated a developmentally regulated expression of the mRNAs for FGFR-1, FGFR-3, FGFR-4, whereas only traces of FGFR-2 mRNA were found. Each receptor type had a different developmental pattern, suggesting an independent regulation. Signal for FGFR-3 was retained only in the adult muscle. Among the low-affinity FGF binding sites, perlecan was absent, whereas RNA transcript for syndecan-1 peaked at week 13 of gestation, after which a significant decrease was observed. Immunohistochemistry for FGFRs revealed that their localization changed with muscle maturation. At early embryonic stages, FGFR-3 and FGFR-4 had a scattered distribution in the tissue, and FGFR-1 was found on myotube and myofiber plasma membranes. At later stages, FGFR-1 positivity decreased and was found in a few areas of the muscle, FGFR-3 was concentrated in the nuclei of some, but not all, muscle fibers, and FGFR-4 maintained an association with plasma membrane. In adult tissue, weak positivity for FGFR-3 and FGFR-4 was observed in the connective tissue only. When immunocytochemistry was performed on human fetal myoblasts in culture, confocal microscope analysis revealed a nonhomogeneous cell membrane distribution of FGFRs. Taken together, the data strongly suggest that developmentally regulated expression and cell distribution of FGFRs play a role during muscle maturation.

Effect of dystrophin antisense oligonucleotides on cultured human neurons.

Antisense oligonucleotides offer the potential to block the expression of specific molecules within the cell, thus providing a useful tool in cell function studies. In this paper, we tested the possibility to block dystrophin expression in in vitro cultured neurons with antisense oligonucleotides administration. Human fetal neuronal cultures were treated with different doses of antisense oligonucleotides against dystrophin, the protein coded by the Duchenne muscular dystrophy gene. Results showed that labelled oligonucleotides rapidly accumulated into cultured neurons, but were discarded 15-24 h after treatment. However, no effects could be observed until 3-4 days after treatment, when immunocytochemical staining for dystrophin was significantly decreased in treated neurons. This result was confirmed by polymerase chain reaction assay which showed a significantly lower expression of the dystrophin specific mRNA. Electron microscope observations confirmed that neurons were affected. Large inclusions or packed granules were detectable in their cytoplasm and in terminal endings. Neuronal nuclear membrane was sometimes shredded, so that nuclear shape was altered. These phenomena were dose-dependent, further substantiating the hypothesis of a specific effect of antisense treatment. This interpretation was supported by the absence of alterations when cultures were treated with mismatch or non specific antisenses. Since the function of dystrophin is still unknown, these data might help in understanding the role played by this protein in the developing brain.

Exon/intron structure of the human AF-4 gene, a member of the AF-4/LAF-4/FMR-2 gene family coding for a nuclear protein with structural alterations in acute leukaemia.

The AF-4 gene on human chromosome 4q21 is involved in reciprocal translocations to the ALL-1 gene on chromosome 11q23, which are associated with acute lymphoblastic leukaemias. A set of recombinant phage carrying genomic fragments for the coding region and flanking sequences of the AF-4 gene were isolated. Phage inserts were assembled into four contigs with 21 exons, and an intron phase map was produced enabling the interpretation of translocation-generated fusion proteins. The gene contains two alternative first exons, 1a and 1b, both including a translation initiation codon. The translocation breakpoint cluster region is flanked by exons 3 and 6 and two different polyadenylation signals were identified. Polyclonal antisera directed against three different portions of the AF-4 protein were produced and used to detect a 116 kD protein in cellular extracts of human B-lymphoblastoid and proB cell lines. In mitogen-stimulated human peripheral blood mononuclear cells the AF-4 antigen was predominantly located in the nucleus. The AF-4 gene is a member of the AF-4, LAF-4 and FMR-2 gene family. The members of this family encode serine-proline-rich proteins with properties of nuclear transcription factors. Comparison of AF-4 protein coding sequences with the LAF-4 and FMR-2 sequences revealed five highly conserved domains of potential functional relevance.

The human ALL-1/MLL/HRX antigen is predominantly localized in the nucleus of resting and proliferating peripheral blood mononuclear cells.

The ALL-1 gene is an important regulator of embryonal and hematopoietic development, and structural variants of the human gene generated by chromosomal translocations and other genomic alterations presumably act as oncogenes in the pathogenesis of acute leukemias and other hematological malignancies. Antisera against two different epitopes of the human ALL-1 protein (anti-ALL1-N and anti-ALL1-C) were produced. Both sera revealed indistinguishable patterns of antigen localization in human peripheral blood mononuclear cells (PBMCs). In resting PBMCs, the antigen was distributed in a speckled pattern across the nuclei, with an increased density at the nuclear envelope and the nuclear indentation. In mitotically stimulated PBMCs, the antigen surrounded the condensing chromosomes but did not colocalize with chromatin or the nuclear scaffold. The antigen is considered a marker for a novel nuclear subcompartment, a perichromosomal area termed the "chromosomal envelope." In Western blot experiments, the anti-ALL1-N serum reacted with a polypeptide corresponding to the expected full-length 430-kDa ALL-1 protein. Recombinant proteins representing the AT-hook and zinc binding subdomains of the ALL-1 protein interacted in vitro with a degenerate mixture of double-stranded oligodeoxynucleotides. Thus, the ALL-1 protein probably is a DNA-binding protein with both a sequence-unspecific (AT-hook) and a sequence-specific (zinc binding subdomains) double-stranded DNA binding mode.

Modulation of the alpha 2 macroglobulin receptor/low density lipoprotein receptor related protein by interferon-gamma in human astroglial cells.

Alpha 2 macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 Mr/LRP) is a multi-functional cell surface receptor that has been implicated in important processes, such as atherogenesis, cellular migration, immune response and degenerative diseases. Its expression increases in human brain during Alzheimer's disease, tissue injury and neoplastic transformation. In the present paper we studied the regulation of alpha 2 Mr expression by interferon-gamma (IFN gamma) in human astrocytoma cell lines and in fetal astrocytes. Western blots demonstrated an increase of the alpha 2 Mr expression after 24 h of IFN gamma treatment. This effect paralleled the up-regulation of alpha 2 Mr mRNA, as detected by PCR. By prolonging incubation with IFN gamma, we observed a decrement of alpha 2 Mr in IFN gamma treated cells, both by western blot and cytometric analysis. Since in the same cells IFN gamma also up-regulates alpha 2 macroglobulin, this effect may be due to an augmented degradation of the receptor during its recycling.

Basic fibroblast growth factor modulates in vitro differentiation of human fetal microglia.

The effects of basic fibroblast growth factor (bFGF) on differentiation of human fetal microglial cells were investigated. Human ramified microglial cells treated with human recombinant bFGF underwent a morphological change which resulted in a round-shape phenotype. bFGF was also able to induce a dose- and time-dependent increase in cell proliferation and enhanced phagocytic and non-specific esterase activity. These results indicate that ramified microglia, when properly stimulated, are regulated in their physiological and proliferative activities and are transformed into amoeboid forms. Growth factors, such as bFGF, are likely to play a key role in microglial transformation in both normal developing brain and in central nervous system injury.

Expression of MHC class I and class II antigens in pancreatic adenocarcinomas.

The antigens encoded by the major histocompatibility complex (MHC) are cell surface glycoproteins that play a fundamental role in the regulation of the immune response. Anomalous MHC expression in tumor cells has been viewed as an important feature to escape tumor recognition by immune cells. Low or absent MHC class I expression as well as ectopic MHC class II expression have been often observed to correlate with high grade malignancy and metastatic potential in a variety of human cancers. To date, very little investigation of MHC (HLA in man) class I and class II expression in human pancreatic cancer has been reported. We investigated this aspect on frozen sections of 8 pancreatic adenocarcinomas and 18 established in vitro cell lines. HLA class I was expressed in all but two cancers whereas de novo HLA class II expression was detected in 3 of 8 cancers. Interestingly, a hierarchy in the expression of the various subsets of HLA class II was found with HLA- DR > -DP > -DQ. Results on cell lines strongly resembled the ones obtained in cancer tissues. However, a peculiar feature was observed in certain cell lines. HLA class II antigens were expressed in only a few cell lines and in some of them a mixed population of positive and negative cells was found. Sorting and cloning of the two populations confirmed the existence of tumor cell clones with stable and distinct HLA class II phenotype. Taken together, these results indicate the cellular heterogeneity of pancreatic cancer cells with regard to the qualitative and quantitative expression of major histocompatibility complex genes, and may provide new insights for a better understanding of the tumorhost relationships in this extremely severe form of neoplasia.

Presence of pituitary adenylate cyclase-activating polypeptide receptors in Y-79 human retinoblastoma cells.

Cytochemical analysis demonstrated that a high percentage of human Y-79 retinoblastoma cells displayed a specific labeling by the biotinyl derivative of pituitary adenylate cyclase-activating polypeptide (PACAP), a novel neuropeptide of the secretin-vasoactive intestinal peptide (VIP) family of peptides. In cell membranes, the two molecular forms of PACAP, the one with 38 (PACAP 38) and the other with 27 (PACAP 27) amino acids, displaced the binding of 125I-PACAP 27 with IC50 values in the picomolar range and increased adenylyl cyclase activity by 100-fold with EC50 values of 27 and 180 pM, respectively. VIP, human peptide histidine-isoleucine, glucagon, and secretin were much less effective and potent in both receptor assays. The PACAP receptor antagonists PACAP 6-27 and PACAP 6-38 and an antiserum directed against the stimulatory G protein Gs inhibited the PACAP stimulation of adenylyl cyclase. In intact cells, both PACAPs and VIP failed to stimulate the phosphoinositide hydrolysis, whereas in cell membranes PACAP 38, but not the other peptides, produced a modest increase (40%) of inositol phosphate formation with an EC50 value of 22 nM. However, this effect was not antagonized by either PACAP 6-38 or PACAP 6-27. These data demonstrate the presence in human Y-79 retinoblastoma cells of specific PACAP receptors and provide further evidence that PACAP may act as a neurotransmitter/neuromodulator in mammalian retina.

On the presence of DNA polymerase alpha in human lymphocyte nuclei and chromosomes.

Experiments were carried out to correlate the cytological localization of DNA polymerase alpha with the presence of its specific mRNA in human lymphocytes studied at different times after phytohaemagglutinin stimulation. Our data indicated that in resting cells it is not possible to detect DNA polymerase alpha protein or mRNA by Northern hybridization. By contrast, in stimulated cells the detection of mRNA specific for DNA polymerase alpha synthesis is possible after 16 h phytohaemagglutin stimulation, whereas immunolocalization is possible after only 4 h stimulation. Observation of cytological preparations from cells stimulated for times long enough to obtain mitoses surprisingly showed an intense immunoreaction in mitotic chromosomes treated with monoclonal antibodies to DNA polymerase alpha.

Human microglia cultures: a powerful model to study their origin and immunoreactive capacity.

In this paper, we report that pure cultures of human microglia were obtained from long-term astrocytic cultures of human fetal brain. After five to six months and repeated cell passages, macrophage-like cells started to spontaneously form in vitro, so that in two to three weeks the whole culture was populated by them. These cells were grown up to over 50 passages in culture and analyzed for morphology, specific marker positivity, growth rate and major histocompatibility complex (MHC) antigen expression with or without gamma-interferon (IFN) stimulation. We found that, regardless of embryonic age of original cultures (10-15 weeks of gestation), cultures showed a remarkable homogeneity and purity and over 90 stained for typical microglial markers. Under basal conditions, two cell subpopulations similar to those described in vivo, we observed: the reactive 'ameboid' type and the resting 'ramified' one, the latter increasing with time in vitro and cell passages. Both cell subpopulations were capable of active phagocytosis and of high-rate proliferation. They spontaneously expressed low levels of MHC class II antigens, but were negative for MHC class I. Stimulation with gamma-interferon lymphokine upregulated the MHC class II expression as well as the MHC class I heavy chain form in ameboid, 'reactive' cells but not in the ramified ones. We also found that beta 2 microglobulin, already expressed in basal conditions, was dissociated from HLA A-B-C molecules in lymphokine-stimulated cells at early passages. The physiological significance of these data, as well as the possible correlation with in vivo ontogenetic modifications, are also discussed.

Expression of basic fibroblast growth factor and its receptors in human fetal microglia cells.

The presence of basic fibroblast growth factor (bFGF) and FGF receptors was investigated in microglia cells derived from human fetal brain long-term cultures. Production of bFGF was suggested through the capability of microglial extracts to stimulate plasminogen activator (PA) synthesis in endothelial cells. The identity of PA-stimulating activity with bFGF was confirmed by its high affinity for heparin and its cross-reactivity with polyclonal antibodies to human recombinant bFGF. These antibodies recognized a cell-associated M(r) 18,000 protein as well as trace amounts of the M(r) 24,000 bFGF isoform in Western blot. All microglial cells showed bFGF immunoreactivity in the cytoplasm and, sometimes, in the nucleus. Scatchard plot analysis of 125I-bFGF binding data revealed the presence of low affinity heparansulphate proteoglycans (380,000 +/- 60,000 sites/cell; Kd = 730 +/- 200 nM) and of high affinity tyrosine-kinase receptors (10,300 + 2500 sites/cell; Kd = 30 +/- 9 pM). Immunocytochemistry confirmed the presence of FGF receptor (1/flg) on the cell surface of some, but not all microglial cells, with prevalent association to ameboid microglia. Transcripts for FGF receptors 1, 2, 3 and 4 were found in microglia by Northern blot analysis. Co-expression of bFGF and its receptors in human fetal microglia suggests an autocrine role of bFGF in these cells.

Immunolocalisation of dystrophin in the immature human neurons and muscles.

Dystrophin, the product of the Duchenne muscular dystrophy gene, has been shown to be developmentally regulated in both human muscle and brain tissues. We consequently performed an immunocytochemical study using electron microscopy to localise the protein in the immature human fetal muscle and neurons. Results demonstrated that, even if dystrophin was partially associated to the plasma membrane in both tissues, some product was also linked to the neurofilaments network in neurons and to microfilaments in muscle. An intense staining was also found in satellite cells.