Effects of developmental lead exposure on the hippocampal methylome: Influences of sex and timing and level of exposure.

Developmental lead (Pb) exposure results in persistent cognitive/behavioral impairments as well as an elevated risk for developing a variety of diseases in later life. Environmental exposures during development can result in a variety of epigenetic changes, including alterations in DNA methylation, that can influence gene expression patterns and affect the function and development of the nervous system. The present promoter-based methylation microarray profiling study explored the extent to which developmental Pb exposure may modify the methylome of a brain region, hippocampus, known to be sensitive to the effects of Pb exposure. Male and female Long Evans rats were exposed to 0 ppm, 150 ppm, 375 ppm, or 750 ppm Pb through perinatal exposures (gestation through lactation), early postnatal exposures (birth through weaning), or long-term postnatal exposures (birth through postnatal day 55). Results showed a significant contribution of sex to the hippocampal methylome and effects of Pb exposure level, with non-linear dose response effects on methylation. Surprisingly, the developmental period of exposure contributed only a small amount of variance to the overall data and gene ontology (GO) analysis revealed the largest number of overrepresented GO terms in the groups with the lowest level of exposure. The highest number of significant differentially methylated regions was found in females exposed to Pb at the lowest exposure level. Our data reinforce the significant effect that low level Pb exposure may have on gene-specific DNA methylation patterns in brain and that this occurs in a sex-dependent manner.

Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II Binding.

Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an antagonist of CCR5 coreceptor activity. These findings suggest that the molecular anatomy of CCR5 binding plays a critical role in antagonism of coreceptor activity.

Identification of ENV determinants in V3 that influence the molecular anatomy of CCR5 utilization.

The V3 loop of the ENV glycoprotein exerts a dominant influence on the interaction of gp120 with coreceptors. Primary env genes cloned from sequential isolates from two seroconverters revealed Pro-->Ala conversion in the conserved GPG motif of the V3 crown in seven of 17 R5 ENV. ENV containing the GPG motif in the V3 crown had fusogenic activity with chimeric receptors containing either the N terminus or loops of CCR5, whereas those with the GAG variant utilized only the former. Site-directed mutagenesis of multiple primary and prototypic R5 env genes demonstrated that the GPG motif was necessary for dual utilization of the N terminus and body of CCR5 in both gain and loss-of-function experiments. All ENV containing the GPG V3 crown showed CCR5 binding in the presence of soluble CD4, whereas it was not detected with the GAG variants. Molecular dynamic simulations of a V3 peptide predicts that the Pro-->Ala substitution results in a conformational change with loss of the crown structure. These studies demonstrate that sequences in the third hypervariable region determine the specificity of coreceptor utilization for fusion, and that a conserved motif in the crown directly influences the molecular anatomy of the interaction between gp120 and CCR5.

Flow cytometric monitoring of red blood cell chimerism after bone marrow transplantation.

Chimerism after bone marrow transplantation (BMT) was investigated by flow cytometry analysis of red blood cells (RBCs) and of reticulocytes using a series of selected monoclonal or polyclonal antibodies directed against ABO, Rhesus, Kell, Duffy or MNSs antigens. The method allows the routine detection of less than 0.1% of positive cells in artificial mixed field populations. Blood samples from 135 patients undergoing BMT were investigated around days 15, 30, 45, 60, 90, 180, and then every 6 months after transplantation. Characteristic patterns showing expression of donor red blood cell antigens (expansion markers) and concomitant decrease of recipient specific antigens (depletion markers) within days 16-20 were observed for 125 successfully engrafted patients. Distinct patterns were obtained in 10 patients. A delay in engraftment was evidenced in four patients by the absence of chimerism during the first 6 months without any sign of relapse. Re-appearance of recipient RBCs and reticulocytes was observed in five patients; it was consistent with relapse that was later confirmed by clinical, haematological and cytogenetic studies. Finally, a stable and partial chimerism with 20% of RBCs expressing a marker from the recipient was observed in one patient without any sign of relapse. The reported investigation demonstrated that flow cytometry of RBCs and reticulocytes represents a powerful method to efficiently monitor bone marrow transplanted patients on a long-term basis.

New method for quantitative determination of fetal hemoglobin-containing red blood cells by flow cytometry: application to sickle-cell disease.

A new method to quantify red blood cells (RBCs) containing fetal hemoglobin (HbF), or F cells, by flow cytometry was developed. The use of formaldehyde as fixative agent and sodium dodecyl sulfate (SDS) to permeabilize fixed RBCs resulted in a low staining background, a negligible Hb leakage, and minimal cell clumping. HbF was detected by immunofluorescence with a murine monoclonal antibody directed to the gamma chain of human Hb. The accuracy of the F-cell count was evaluated on mixed-field populations of adult and cord RBCs. The results of flow cytometry were highly correlated with HbF dosage in the hemolysate by high-pressure liquid chromatography (HPLC) (R > 0.99). The sensitivity of the method allowed the study of HbF distribution at the single-cell level in normal controls and patients with sickle-cell disease (SCD). All patients exhibited a heterocellular distribution of HbF, with highly variable percentages of F cells and non-F cells. As an additional and valuable biological parameter in SCD, the mean HbF content per F cell could be deduced from the measurement of HbF level by HPLC and F-cell count by flow cytometry. Results were unchanged after storing the cells for several weeks in an optimized resuspending solution. Since it does not need uncommon reagents or devices and allows the simultaneous detection of membrane antigens by two-color flow cytometry, this method is convenient for routine laboratories as well as for research purposes.

X-linked vacuolated myopathy: membrane attack complex deposition on the surface membrane of injured muscle fibers is not accompanied by S-protein.

We have studied the expression of S-protein on the muscle from patients with X-linked vacuolated myopathy [characterized by the deposition of the complement C5b-9 membrane attack complex (MAC) over abnormal muscle fibers] and controls by immunocytochemistry and immunoblotting. No expression was detected on muscle from controls and patients with X-linked vacuolated myopathy. These findings suggest that S-protein does not render the MAC inactive in X-linked vacuolated myopathy. This situation may be due to the fact that the pathways of MAC activation and the expression of S-protein in X-linked vacuolated myopathy are different from the ones observed in ischemic and/or necrotic, or immune diseases. These results emphasize the role of the membrane complement regulatory proteins (i.e., CD59) in X-linked vacuolated myopathy.

Investigation of the survival of paroxysmal nocturnal hemoglobinuria red cells through the immunophenotyping of reticulocytes.

BACKGROUND: Complement-mediated lysis of red cells (RBCs) is a classic feature of paroxysmal nocturnal hemoglobinuria (PNH) that is traditionally studied with a combination of radiolabeling of RBCs and in vitro hemolysis tests. Phenotyping of reticulocytes was used as an alternative method for the evaluation of the relative life span of normal RBCs (PNH I) and RBCs that were partially (PNH II) or completely (PNH III) deficient in CD59. STUDY DESIGN AND METHODS: Murine monoclonal antibodies CD55, CD58, and CD59 and thiazole orange were used to phenotype reticulocytes by two-color flow cytometry in nine PNH patients. RBC survival could be calculated from the ratio of CD59- or CD59low mature RBCs to CD59- or CD59low reticulocytes obtained from these patients who had not received a transfusion. RESULTS: The life span of PNH III RBCs varied from about 17 to 60 days. PNH II reticulocytes were found in the four patients with PNH II RBCs. The life span of PNH II RBCs varied with their residual expression of CD59, and cells with 15 to 20 percent of the normal amount of CD59 were protected against in vivo hemolysis. CONCLUSION: Phenotyping of reticulocytes is a convenient and reliable tool for evaluating the relative survival of normal and PNH RBCs. PNH II and PNH III reticulocytes are phenotypically distinct, and some PNH II RBCs may be sensitive to complement-mediated lysis in vitro, but normally they are complement-resistant in vivo.

Expression of blood group i antigen and fetal hemoglobin in paroxysmal nocturnal hemoglobinuria.

BACKGROUND: The mechanism by which the paroxysmal nocturnal hemoglobinuria (PNH) clone progressively takes over normal hematopoietic cells remains unknown. The respective in vivo differentiation of normal and PNH erythroid progenitors was investigated through the expression of two fetal erythroid markers (i antigen and fetal hemoglobin [HbF]) whose expression in adult red cells is associated with altered erythropoiesis. STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against HbF and i and CD59 antigens were used to phenotype red cells of 10 PNH patients. A multiparametric flow cytometry assay of red cells and reticulocytes was designed to assess a possible association of i and HbF with PNH or normal red cells. RESULTS: Most patients exhibited greater expression of i and HbF than did normal controls. In each case, the percentages of i-positive or HbF-positive cells within CD59-deficient and CD59-positive red cells were very close, clearly showing a lack of preferential association of these markers with normal or PNH cells. CONCLUSION: In PNH patients, normal and PNH erythroid progenitors have the same ability to promote HbF and i antigen expression, which suggests that normal and PNH erythroid progenitors (burst-forming units-erythroid, colony-forming units-erythroid, erythroblasts) behave similarly in response to bone marrow stress.

Flow cytometry and immunoblotting analysis of monoclonal antibodies directed to complement regulatory proteins.

A series of 18 monoclonal antibodies directed to complement regulatory proteins were investigated by flow cytometry and immunoblotting. Seventeen antibodies are directed against a phosphatidyl inositol-linked glycoprotein since they show a dual population of erythrocytes from a paroxysmal nocturnal haemoglobinuria (PNH) patient. From this group, 6 antibodies revealed a 65-70 kDa band on immunoblots allowing their identification as anti-DAF (Decay Accelerating Factor; CD55 antigen), and 11 bound to a 20 kDa molecule corresponding to MIRL (Membrane Inhibitor of Reactive lysis, CD59 antigen). One antibody revealed an homogeneous population from the PNH patient and bound to a 200 kDa band on immunoblot that might corresponds to the CR1 (Complement Receptor type 1; CD35).

Expression of CD59, a regulator of the membrane attack complex of complement, on human skeletal muscle fibers.

Control of complement deposition on autologous cells is mediated by a group of complement regulatory membrane proteins acting at different levels of the complement cascade. Decay accelerating factor (CD55) prevents the assembly of C3 convertases and CD59 membrane inhibitor of reactive lysis (MIRL) restricts homologous complement lysis by the membrane attack complex of complement (MAC) by inhibition of C5b-8 catalyzed insertion of C9. The aim of this work was to study the eventual expression of CD55 and CD59 on human skeletal muscle fibers. Highly sensitive immunoblotting using murine monoclonal antibodies showed that CD59, but not CD55, was present in skeletal muscle fibers. Immunocytochemistry with a monoclonal antibody against CD59 demonstrated a dense granular immunostaining mainly localized at the level of the sarcolemma. Thus, CD59, but not CD55, is expressed on normal skeletal muscle fibers. CD59 may play a prominent role in preventing MAC deposition and subsequent complement-mediated damage in myopathies where the complement system activation is involved.

Expression of glycosyl-phosphatidylinositol-linked glycoproteins in blood cells from paroxysmal nocturnal haemoglobinuria patients: a flow cytometry study using CD55, CD58 and CD59 monoclonal antibodies.

We used CD55 and CD59 monoclonal antibodies (mAbs) for the investigation of peripheral blood cells from sixteen patients with paroxysmal nocturnal haemoglobinuria (PNH) by flow cytometry. Mixed-field populations of erythrocytes, platelets, monocytes or granulocytes were visualized in all cases but, due to the extended half-life of unaltered erythrocytes and blood transfusions, the GPI-linked protein deficiency was more obvious for white blood cells, especially granulocytes, and platelets. In contrast, a minority of altered lymphocytes was observed in only five patients. The expression of CD55 and CD59 antigens was grossly correlated but quantitative differences were observed for patients whose cells were only partially deficient in GPI-linked proteins. Patients with PNH secondary to aplastic anaemia exhibited a significantly lower percentage of altered cells compared with "classical" PNH patients. In addition, we found that CD58 normally bound to deficient white blood cells and platelets, showing the LFA-3 molecule is expressed as a transmembrane protein in these cells.

Collagen matrix: an attractive alternative to agar and methylcellulose for the culture of hematopoietic progenitors in autologous transplantation products.

Autografts using untreated or in vitro manipulated bone marrow and peripheral blood stem cells represent promising approaches to the treatment of malignant diseases. In this work, the collagen gel culture technique was compared with agar and methylcellulose for its capacity to permit the growth of human granulomonocytic (day 14 CFU-GM; collagen vs agar or MTC) or erythroblastic (day 7 CFU-E and day 14 BFU-E; collagen versus methylcellulose) colonies in autologous transplantation products. Our results show that the collagen culture system always gave as many or more colonies than the other techniques. It also allowed harvesting of gels onto glass slides and subsequent May-Grünwald-Giemsa, cytochemical or immunocytochemical staining. We suggest that the collagen assay represents an interesting alternative to the widely used agar or methylcellulose systems for the culture of hematopoietic progenitors because of the equal or higher number of colonies detected, the easy phenotypical identification of colonies in stained gels, and the ability to store high-quality documentation. This technique is particularly attractive for use in the quality control of autologous bone marrow transplantation procedures.

Protection of rat endothelial cells from primate complement-mediated lysis by expression of human CD59 and/or decay-accelerating factor.

The present study analyzed the ability of human decay-accelerating factor (DAF) and CD59 to protect rat endothelial cell (EC) clones from human and primate complement-mediated lysis. By flow cytometry and Scatchard analysis, we show that human DAF and/or CD59 cDNAs under the transcriptional control of elongation factor 1-alpha promoter were expressed at levels similar to or higher than that of a human EC line. Human DAF and CD59 were released from the surface of transfected rat cells by phosphatidylinositol phospholipase C, demonstrating that the two molecules were linked to the cell membrane by means of a glycolipid anchor. The functional activity of the two human C regulatory proteins expressed on rat EC lines was studied using an in vitro assay of C-dependent cytotoxic in which rat EC were incubated with human or nonhuman primate sera as sources of xenogeneic natural antibodies and C. We demonstrate that human and monkey xenogeneic natural antibodies bind to rat cells and induce lysis by a C-dependent mechanism involving mainly the C direct activation pathway. Our data indicate that human DAF and CD59, expressed either alone or in combination, abrogated all EC cytotoxicity, even in the presence of 50% human serum. This protective phenotype was correlated with decreased membrane attack complex (CD59 and/or DAF transfectants) and C3 deposition (DAF transfectants) on EC surface. Antibodies against the transfected molecules abolished the protection against C-mediated lysis.

Production of a new murine monoclonal antibody with Fy6 specificity and characterization of the immunopurified N-glycosylated Duffy-active molecule.

A murine monoclonal antibody (mAb i3A; IgG1, kappa light chain) was obtained using human red blood cells as immunogen. The antibody showed Fy6 specificity since it agglutinated all but Fy(a-b-)-untreated red cells and failed to agglutinate chymotrypsin-treated cells. An erythrocyte membrane protein of 42-46 kD was revealed as the major component recognized by the antibody on immunoblots. The antibody also bound to 92- to 95- and 200-kD proteins, tentatively identified as oligomers of the 42- to 46-kD monomeric form. The affinity-purified Fy6-active protein was converted to a sharp band of 35 kD after N-glycanase treatment. The molecule appeared as a slightly broadly band after neuraminidase treatment but was not further altered by O-glycanase. The i3A mAb bound to 6,000 +/- 1,000 receptor sites on either Fy(a-b+), Fy (a+b+) and Fy(a+b-) red cells with an affinity constant in the range of 3-6 x 10(8) M-1. No binding was observed to other blood cells nor to several cells (B, T, myelomonocytic and erythro-leukemia cell lines). Also, the bulk of i3A-Fy6 immune complexes could be dissociated from the red cell membrane with as low as 0.2% Triton X-100, showing that the Fy6-active glycoprotein is not tightly associated with the membrane skeleton. Our data obtained with a new monoclonal antibody directed to the Fy6 antigen demonstrate that the blood group Duffy-active component is a red cell-specific glycoprotein carrying one or more N-linked oligosaccharides.

[Rapid diagnosis of paroxysmal nocturnal hemoglobinuria by gel test agglutination]. / Diagnostic rapide des hémoglobinuries nocturnes paroxystiques par agglutination en gel.

Murine monoclonal antibodies (MoAbs) directed against DAF (Decay Accelerating Factor, CD55 antigen) and MIRL (Membrane Inhibitor of Reactive Lysis, CD59 antigen) were used to identify the affected red cells (CD55-/CD59-) of PNH patients. MoAbs NaM16-4D3 (CD55, IgG2a) and NaM77-1E5 (CD59, IgG3) weakly agglutinate red cells and represent powerful tools to quantitate normal (PNHI) and abnormal (PNHII and PNHIII) cells from PNH patients by indirect flow cytometry. MoAbs NaM125-7H10 (CD55) and NaM123-6G12 (CD59), both IgM, were selected for their agglutinating properties and used for the separation of PNHI from PNHII and PNHIII red cells by the gel test technology. From analysis of artificial mixtures of DAF+ and DAF- cells, a direct relationship was established between fluorescent cells detected by flow cytometry, and erythrocytes agglutinated in microtyping cards. The method was further confirmed by analysis of ten blood samples from PHN patients and represent an alternative to classical hemolysis tests. On the basis of our experience we propose the following for the diagnosis of PNH: 1) agglutination test with NaCl microtyping cards using IgM CD55 and CD59; 2) flow cytometry analysis for accurate quantitation of CD55-/CD59- red cells.