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.

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.

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.

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.

Surface markers on lymphocytes from human cerebrospinal fluid. Identification by monoclonal antibodies.

Lymphocyte subpopulations in cerebrospinal fluid (CSF) and peripheral blood (PB) were studied using monoclonal antibodies and the common membrane markers. The results in three groups of patients were compared: 36 subjects with 'non-immunological disorders' (NID), 14 subjects with multiple sclerosis (MS) and 6 with subacute sclerosing panencephalitis (SSPE). It was found that, in patients with NID, (1) 90% of cells were T lymphocytes, reactive with OKT3; (2) the helper/suppressor (T4/T8) ratios were the same in the CSF and the PB; (3) the OKIa1 percentage was lower in the CSF than in the PB; and (4) only a few cells were 'immature', reacting with OKT10. Using the membrane markers (E rosettes, Fc IgG receptors and surface immunoglobulins), on the other hand, it was noted that the majority of cells in the CSF were identified as suppressor T lymphocytes and surface immunoglobulin-positive B cells were less common than the Ia1 marker suggested. There were no significant differences between the CSF results in patients with NID and MS but the OKT3 lymphocytes were reduced in CSF samples from patients with SSPE.

Lymphocyte subsets in human adenoids and tonsils. Rosette formation, alpha-naphthyl acetate esterase cytochemistry, monoclonal antibodies and peanut lectin reactivity.

The distribution of mononuclear cell subsets has been studied in human adenoids, tonsils and peripheral blood (PB) by evaluating the presence of surface immunoglobulins, E-rosette formation, receptors for IgG Fc and for complement, alpha-naphthyl acetate esterase (ANAE) cytochemistry, reactivity with peanut lectin (PNA) and with monoclonal antibodies (McAb) (OK panel). Adenoids and tonsils, compared to PB, contain (1) fewer macrophages and T cells but more B cells; (2) higher proportions of ANAE negative, complement receptors and Ia-like antigens bearing T lymphocytes; (3) higher percentages of cells reacting with the McAbs OKT9 and OKT10 ("immature" lymphoid cells). In both adenoids and tonsils, clusters, formed by a central heavily ANAE stained interdigitating cell surrounded by lymphocytes with a sickle-shaped ANAE reaction, were found. Analogous clusters have been previously described in mice and human thymus. Two major hypotheses could be put forward: (1) adenoids and tonsils contain "immature" lymphoid cells undergoing education process, or (2) the above organs contain lymphocytes activated by a constant exposure to bacterial antigens or mitogens.

Infantile cortical hyperostosis (Caffey disease): ultrastructural and immunohistochemical characterization of the peritrabecular cells.

The ultrastructure and the immunohistochemical pattern of the cells which are responsible for the bone resorption in the cortical infantile hyperostosis were investigated. The osteoclasts present a great positivity to MB1 antigen and a low positivity to OKM5. Mononuclear cells with primary lysosomes, looking like osteoclast ones are present in high concentration in peritrabecular spaces. These cells show a high positivity to OKM5 antigen and a low positivity to MB1 antigen. The mononuclear granulated cells are positive to tartrate-resistent acid phosphatase. The possible common origin and their co-operation in bone resorption is discussed.

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.

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.

Differential expression of fibroblast growth factor receptors by human neurones, astrocytes and microglia.

Expression of fibroblast-growth factor receptors (FGFRs) was studied in human fetal neurones, astrocytes and microglia in culture. Northern blot analysis showed that neurones and microglia expressed the mRNAs for FGFR-1, FGFR-2, FGFR-3, FGFR-4 at different levels, whereas astrocytes expressed only FGFR-1 and FGFR-4 mRNAs. Immunocytochemical localization of FGFR-1 revealed that this receptor was predominantly localized on the axon hillock membrane in neurones, and was associated with the plasma membrane of ameboid, activated microglia and of glial-fibrillar acidic protein positive astrocytes. The expression of various members of the FGFR family in all the cell types investigated implicates FGFs in human brain development and functions.

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.

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.

Antigenic and cytochemical characterization of cells adhering to intrauterine contraceptive devices.

Eighteen copper intrauterine devices (IUDs), removed after 25 months of use, were examined to evaluate cells adhering to them (IAC). By means of monoclonal antibodies, the antigenic phenotype of IAC was studied, along with some IAC cytochemical properties. Sixty percent of IAC were identified as granulocytes based on morphological, cytochemical, and antigenic characteristics. A small proportion of IAC were shaped like large foreign-body macrophages, with multiple picnotic nuclei, and diffused alpha naphthyl acetate esterase (ANAE) activity. Some IAC identified as macrophages from a morphological view point also showed dipeptidyl-diaminopeptidase IV (DAPIV) reactivity, previously described only in T-helper lymphocytes. Most IAC identified as macrophages reacted with the monoclonal antibodies OKM1 and HLA-DR, and showed ANAE activity in the form of small multiple granules. The hypothesis that IUD-adhering macrophages with an ANAE+, DAPIV+, OKM1+, and HLA-DR+ phenotype could play a role in the inactivation of spermatozoa can be proposed.

Lymphocyte subsets at different stages of subacute sclerosing panencephalitis: a study with monoclonal antibodies.

Lymphocyte subsets in cerebrospinal fluid (CSF) and peripheral blood were studied using monoclonal antibodies, in patients with subacute sclerosing panencephalitis, eight of whom were at stage 2 and seven at stage 4. Eighteen subjects affected with non immunological diseases constituted the controls. Regardless of the stage, patients with subacute sclerosing panencephalitis had lower percentages of OKT3+ (pan-T) cells in both CSF and peripheral blood, with an increase of OKIa+ cells (B cells, macrophages and active T cells) in peripheral blood. A difference was found in the proportion of OKT4+ (helper-inducer) and OKT8+ (suppressor/cytotoxic) cells in relation to the stage, the most striking finding being a significant decrease of OKT8+ with an increase of T4/T8 ratio in peripheral blood at an early stage.

Cerebrospinal fluid lymphocyte subpopulations in multiple sclerosis.

T3+ (all-T) and T8+ (suppressor/cytotoxic) cells were studied in cerebrospinal fluid (CSF) from 24 patients with multiple sclerosis (MS) and from 24 subjects with various "non-immunological" disease (NID). MS patients were classed as (a) during the acute phase of the 1st episode of the disease, (b) in acute relapse, (c) with chronic progressive disease, (d) with increased or (e) normal CSF IgG content or (f) with neurological impairment (Kurtzke scale) less than or equal to 3 or (g) greater than 3. In MS cases considered as a whole a significant decrease in CSF T3+ cells was found compared to NID patients. When single groups were considered, T3+ cells decrease was significant in classes (b), (d) and (f). Significantly lower percentages of T8+ cells, compared to NID, were found in MS classes (a), (d) and (f).