Identification of antibodies directed against O-acetylated sialic acids in visceral leishmaniasis: its diagnostic and prognostic role

A significantly increased O-acetylated sialic acid (O-AcSA) binding fraction was purified from serum of visceral leishmaniasis (VL) patients by affinity chromatography on immobilized bovine submaxillary mucin (BSM) and found to be immunoglobulin in origin. The serodiagnostic and prognostic potential of BSM as a capture antigen was established by ELISA with no cross reactivity with coendemic diseases like malaria, tuberculosis, leprosy, chagas disease and cutaneous leishmaniasis; however, a strong cross reactivity was present with trypanosomiasis patients. In 56 clinically diagnosed VL patients, the BSM-ELISA was compared with diagnosis by microscopy using Giemsa stained tissue smears and direct ELISA using crude parasite antigen (parasite-ELISA); 49/56(87.5%) and 5/56(9.0%) were positive and negative respectively by all 3 methods. The BSM-ELISA failed to diagnose 2/56(3.5%) patients which were biopsy and parasite-ELISA positive. The prognostic potential of the BSM-ELISA in 18 longitudinally monitored VL patients before and after conventional antimonial treatment showed a significant decrease in anti O-AcSA titres in drug responsive patients whereas anti O-AcSA levels persisted in drug unresponsive patients. The IgG subclass distribution of antibodies directed against O-AcSA showed increased IgG2 levels in VL patients as compared to healthy controls. The BSM-based ELISA holds great promise as a serodiagnostic and prognostic assay for VL.     

Interferon gamma promotes survival of lymphoblasts overexpressing 9-O-acetylated sialoglycoconjugates in childhood acute lymphoblastic leukaemia (ALL)

An enhanced linkage-specific 9-O-acetylated sialic acid (9-O-AcSA) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia, ALL (PBMC(ALL), 9-O-AcSA+ cells) was demonstrated by using a lectin, Achatinin-H, whose lectinogenic epitope was 9-O-AcSAalpha2-6GalNAc. Our aim was to evaluate the in vitro contributory role of this glycotope (9-O-AcSAalpha2-6GalNAc) towards the survival of these 9-O-AcSA+ cells in ALL patients. For direct comparison, 9-O-AcSA- cells were generated by removing O-acetyl group of 9-O-AcSA present on PBMC(ALL) using O-acetyl esterase. An elevated level of serum interferon gamma (IFN-gamma) in affected children led us to think that PBMC(ALL) are continuously exposed specifically to this cytokine. Accordingly, 9-O-AcSA+ and 9-O-AcSA- cells were exposed in vitro to IFN-gamma. A twofold increased NO release along with inducible NO synthase (iNOS) mRNA expression by the 9-O-AcSA+ cells was observed as compared to the 9-O-AcSA- cells. The decreased viability of IFN-gamma exposed 9-O-AcSA- cells as compared to 9-O-AcSA+ cells were reflected from a 5.0-fold increased caspase-3-like activity and a 10.0-fold increased apoptosis in the 9-O-AcSA- cells when production of NO was lowered by adding competitive inhibitor of iNOS in reaction mixture. Therefore, it may be envisaged that a link exists between induction of this glycotope and their role in regulating viability of PBMC(ALL). Taken together, it is reasonable to hypothesise that O-acetylation of sialic acids on PBMC(ALL) may be an additional mechanism that promotes the survival of lymphoblasts by avoiding apoptosis via IFN-gamma-induced NO production.     

Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukemia

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-alpha2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac(2)-GPs(ALL) and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac(2)-GPs(ALL) were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac(2)-GP(ALL) antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac(2)-GPs(ALL) as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac(2)-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac(2)-GPs(ALL) as an alternate tool for detection of anti-Neu5,9Ac(2)-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.     

Identification of 9-O-acetylated sialoglycans on peripheral blood mononuclear cells in Indian Visceral Leishmaniasis

Although the existence of O -acetylated sialic acids is well known, it is only in recent years that steady refinement of analytical techniques have enabled detailed mapping of their structural diversity [1]. Fluorimetric analysis of peripheral blood mononuclear cells (PBMC) of patients with Visceral Leishmaniasis (VL) showed six fold increase in the percentage of surface 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) as compared to normal human donors. Using Achatinin-H, a 9-O-acetyl sialic acid- binding lectin, an enhanced presence of 9-O-AcSGs in an alpha2 --> 6 linkage was demonstrated by flow cytometry; abolition of its binding by pretreatment with a recombinant 9-O-acetylesterase corroborated the presence of this glycotope. Western blotting of PBMC from VL patients indicated the presence of five O-acetylated sialoglycans corresponding to 144, 65, 56, 36 and 19 kDa as compared to 144 and 36 kDa in normal individuals. Taken together our data indicates that during active disease, there is an overexpression of 9AcSGs on the surface of PBMC of VL patients, thus opening up new research avenues wherein the expression of this biomarker could be exploited to monitor the clinical status of VL patients.     

Regulation of O-acetylation of sialic acids by sialate-O-acetyltransferase and sialate-O-acetylesterase activities in childhood acute lymphoblastic leukemia

Enhanced expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) and 9-O-acetylated disialoganglioside (9-OAcGD3) was observed on lymphoblasts of childhood acute lymphoblastic leukemia (ALL). Sialate-O-acetyltransferase (SOAT) and sialate-O-acetylesterase (SIAE) are the two main enzymes responsible for the quantity of the O-acetyl ester groups on sialic acids (Sias). We have earlier shown an enhanced level of SOAT activity, capable of transferring acetyl groups to Sias of glycoconjugates in the microsomes of lymphoblasts of these children. We further observed a decreased SIAE activity in both lysosomal and cytosolic fractions of ALL cell lines and primary cells from bone marrow of patients compared with peripheral blood mononuclear cells from healthy donors, which preferentially hydrolyze O-acetyl groups at C-9 of Sia. The level of O-acetylated Sias in the cytosolic and the lysosomal fractions showed a good correlation with SIAE activity in the corresponding fractions. The apparent K(M) values for SIAE in the lysosomal and the cytosolic fractions from lymphoblasts of ALL patients are 0.38 and 0.39?mM, respectively. These studies demonstrate that both SIAE and SOAT activities seem to be responsible for the enhanced level of Neu5,9Ac(2) in lymphoblasts, which is a hallmark in ALL. This was subsequently confirmed by using an enzyme-linked immunosorbent assay that also demonstrated a steady decline in SOAT activities even in cell lysates of lymphoblasts during successful chemotherapy, like radioactive methods have shown earlier.     

O-acetylation of GD3 prevents its apoptotic effect and promotes survival of lymphoblasts in childhood acute lymphoblastic leukaemia

We have previously demonstrated induction of O-acetylated sialoglycoproteins on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). These molecules promote survival of lymphoblasts by preventing apoptosis. Although O-acetylated sialoglycoproteins are over expressed, the status of O-acetylation of gangliosides and their role in lymphoblasts survival remains to be explored in ALL patients. Here, we have observed enhanced levels of 9-O-acetylated GD3 (9-O-AcGD3) in the lymphoblasts of patients and leukaemic cell line versus disialoganglioside GD3 in comparison to the normal cells. Localization of GD3 and 9-O-AcGD3 on mitochondria of patient's lymphoblasts has been demonstrated by immuno-electron microscopy. The exogenous administration of GD3-induced apoptosis in lymphoblasts as evident from the nuclear fragmentation and sub G0/G1 apoptotic peak. In contrast, 9-O-AcGD3 failed to induce such apoptosis. We further explored the mitochondria-dependent pathway triggered during GD3-induced apoptosis in lymphoblasts. GD3 caused a time-dependent depolarization of mitochondrial membrane potential, release of cytochrome c and 7.4- and 8-fold increased in caspase 9 and caspase 3 activity respectively. However, under identical conditions, an equimolar concentration of 9-O-AcGD3 failed to induce similar effects. Interestingly, 9-O-AcGD3 protected the lymphoblasts from GD3-induced apoptosis when administered in equimolar concentrations simultaneously. In situ de-O-acetylation of 9-O-AcGD3 with sodium salicylate restores the GD3-responsiveness to apoptotic signals. Although both GD3 and 9-O-acetyl GD3 localize to mitochondria, these two structurally related molecules may play different roles in ALL-disease biology. Taken together, our results suggest that O-acetylation of GD3, like that of O-acetylated sialoglycoproteins, might be a general strategy adopted by leukaemic blasts towards survival in ALL.     

O-acetyl sialic acid specific IgM in childhood acute lymphoblastic leukaemia

Initial studies have revealed an enhanced surface expression of O-acetylated sialoglycoconjugates (O-AcSGs) on lymphoblasts concomitant with high titres of IgG in childhood Acute Lymphoblastic Leukaemia (ALL) (Mandal C, Chatterjee M, Sinha D, Br J Haematol 110, 801–12, 2000). In our efforts to identify disease specific markers for ALL, we have affinity-purified IgM directed against O-AcSGs that reacts with three disease specific O-AcSGs present on membrane proteins derived from peripheral blood mononuclear cells (PBMC) of ALL patients. Antibody specificity towards O-AcSGs was confirmed by selective binding to erythrocytes bearing surface O-AcSGs, decreased binding with de-O-acetylated BSM and following pretreatment with O-acetyl esterase. Competitive inhibition ELISA demonstrated a higher avidity of IgM for O-AcSG than IgG. Flow cytometry demonstrated the diagnostic potential of purified O-AcSA IgM as binding was specific with ALL patients and minimal with other haematological disorders and normal individuals. It therefore may be adopted as a non-invasive approach for detection of childhood ALL. Taken together, the data indicates that carbohydrate epitopes having terminal O-AcSA 2 6 GalNAc determinants induce disease specific IgG and IgM, potentially useful molecular markers for childhood ALL.     

Lysis of fresh leukemic blasts by interferon-activated human natural killer cells

In a comprehensive study of 30 leukemia patients, it was found that a measurable fraction of fresh leukemic blasts from 8 of 8 adult patients with chronic myelogenous leukemia (CML) in blast crisis and 10 of 11 pediatric patients with childhood acute lymphocytic leukemia (ALL) were efficiently lysed by human peripheral blood natural killer (NK) cells as measured in 4-hour chromium release assays. The observed lysis of these fresh, noncultured, neoplastic blasts was mediated by a population of interferon-augmentable, FcR-positive, non-adherent large granular lymphoid cells from normal donors, which were also able to kill the 'standard' NK target K562. It was of further interest that all 8 of the patients with blast crisis CML exhibited myeloid type morphology. Furthermore, neoplastic lymphoblasts from 9 of 10 patients with NK-susceptible childhood ALL lacked easily detectable B or T cell markers and were of 'null' cell type. In marked contrast to the lytic susceptibility of fresh leukemic blasts from patients with ALL and CML in blast crisis, fresh neoplastic granulocytes from 5 patients with chronic phase CML (2 of which eventually progressed to myeloid type blast crisis), as well as leukemic blasts from 8 patients with acute myeloid leukemias (AML, AMMoL, and AMoL) were resistant to lysis as mediated by human NK cells from normal donors. The clinical implications of these findings are discussed.     

Gram-scale synthesis of pinusolide and evaluation of its antileukemic potential

Pinusolide (1), a known platelet-activating factor (PAF) receptor binding antagonist, was synthesized from lambertianic acid (2), a labdane-type diterpene readily accessible in multigram quantities from the Siberian pine tree. It was shown that 1 not only decreases the proliferation activity of tumor cells at relatively low concentrations but specifically induces apoptosis at 100 microM via the mitochondrial pathway in the Burkitt lymphoma cell line BJAB. Also, using primary lymphoblasts and leukemic cells from children with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), a significant DNA fragmentation in pinusolide-treated cells could be detected in an ex vivo apoptosis assay.     

Identification of 9-O-acetylated sialoglycans on peripheral blood mononuclear cells in Indian Visceral Leishmaniasis

Although the existence of O-acetylated sialic acids is well known, it is only in recent years that steady refinement of analytical techniques have enabled detailed mapping of their structural diversity [1]. Fluorimetric analysis of peripheral blood mononuclear cells (PBMC) of patients with Visceral Leishmaniasis (VL) showed six fold increase in the percentage of surface 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) as compared to normal human donors. Using Achatinin-H, a 9-O-acetyl sialic acid- binding lectin, an enhanced presence of 9-O-AcSGs in an 2 6 linkage was demonstrated by flow cytometry; abolition of its binding by pretreatment with a recombinant 9-O-acetylesterase corroborated the presence of this glycotope. Western blotting of PBMC from VL patients indicated the presence of five O-acetylated sialoglycans corresponding to 144, 65, 56, 36 and 19 kDa as compared to 144 and 36 kDa in normal individuals. Taken together our data indicates that during active disease, there is an overexpression of 9-O-AcSGs on the surface of PBMC of VL patients, thus opening up new research avenues wherein the expression of this biomarker could be exploited to monitor the clinical status of VL patients. Published in 2004..     

Plasma and intracellular levels of lactate dehydrogenase, phosphohexose isomerase and lysozyme activity in acute leukemia

Plasma and intracellular levels of lactate dehydrogenase (LDH), phosphohexose isomerase (PHI) and lysozyme activities were investigated in 20 patients with acute myelocytic leukemia (AML), 18 patients with acute lymphatic leukemia (ALL) and 10 patients with chronic myelocytic leukemia in blast transformation (CML/BT). Though the plasma levels of LDH and PHI in all patients with acute leukemia were elevated as compared to control persons there was no distinctive pattern which could be of use in the classification of acute leukemia. On the other hand the intracellular levels of these enzymes could be of value in classifying acute leukemia. The leukemic lymphoblasts were characterized by low levels of PHI and lysozyme as compared to leukemic myeloblasts or to normal lymphocytes (p less than 0.01). The LDH/PHI ratio is also significantly higher in leukemic lymphoblasts than in leukemic myeloblasts or in normal lymphocytes (p always less than 0.01). These characteristics might also be made use of in identifying the blasts of CML/BT als "lymphoid" or "myeloid" in corresponding cases.     

Antibodies against 9-O-acetylated sialic acids in childhood acute lymphoblastic leukemia: a two-year study with 186 samples following protocol MCP 943

Initial studies have revealed an enhanced surface expression of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) on lymphoblasts concomitant with high titers of antibodies (anti-9-OAcSGs) in childhood acute lymphoblastic leukemia (ALL). This study was undertaken in 186 coded samples from 69 ALL patients to evaluate if antibodies against these sialoglycans could monitor response to the treatment. An ELISA was developed using bovine submaxillary mucin (BSM) containing high % of 9-O-acetylated sialic acids (9-OAcSA) as the capture antigen, to investigate serum levels of anti 9-OAcSGs in a single-center series of pediatric, clinically-diagnosed and immunophenotypically confirmed ALL patients, as compared to 130 healthy controls. At presentation, a 3.8-fold increase in anti-9-OAcSGs levels was detected in 63/69 ALL patients (mean +/- SEM was 102.8 +/- 6.3 microg/ml) as compared to normal controls (27.17 +/- 0.76 microg/ml), assay sensitivity being 91.3%. On an individual basis (n = 25) in patients who were longitudinally monitored for two years, a significant decline in their mean +/- SEM of OD405 was observed from 0.85 +/- 0.06 to 0.28 +/- 0.03. Additionally, a dot-blot was developed to evaluate the proportion of immune-complexed 9-OAcSGs in these patients employing achatinin-H, a 9-OAcSA-binding lectin. Our data indicate that these economically viable ELISA-based approaches allow for reliable, sensitive and rapid diagnosis of ALL. We contend that these disease-specific antibodies could be considered as potential markers both for the initial diagnosis of ALL and possibly for longitudinal monitoring of the disease.     

The detection of Fc-IgA receptors on T and non-T,non-B acute leukemic lymphoblasts

Fc-IgA receptors have been described on purified human T-lymphocyte populations from normal individuals. More recently, non-T-cell subpopulations bearing Fc-IgA receptors have also been described. In this study, receptors for the Fc portion of IgA were detected on both T and non-T, non-B leukemic lymphoblasts from 15 patients with acute lymphoblastic leukemia (ALL). From 1.6 to 6.8% of the T lymphoblasts of 7 patients expressed Fc-IgA receptors and 0.5 to 3.9% of the non-T, non-B lymphoblasts of the remaining 8 patients expressed Fc-IgA receptors. The expression of IgA receptors on these cells may reflect the maturational state of these leukemic lymphoblasts. The detection of Fc-IgA receptors on leukemic lymphoblasts suggests that expression of these receptors on lymphoid cells represents an early maturational event.     

Prooxidant and antioxidant effects of trolox on ferric ion-induced oxidation of erythrocyte membrane lipids

Achatinin_H (ATN_H)is a lectin, isolated from the hemolymph ofAchatina fulica snail, which has been shown to have narrow specificity towards 9-O-acetyl sialic acid. Usually ATN_H does not agglutinate normal human erythrocytes, however, it is capable of agglutinating erythrocytes of patients suffering from acute lymphocytic and acute myelogenous leukemia. Determination of binding constants, numbers of binding sites and lectin overlay experiments using patients' erythrocytes ghost, have suggested that some alterations in erythrocyte cell surface sialoglycoproteins or more precisely appearance of some O-acetylated sialoglycoprotein as a result of pathological transformations has caused this change in the binding of ATN_H.Abbreviations ATN_H Achatinin_H - 9-OAc-NeuAc 9-O-acetyl N-acetyl neuraminic acid - BSM Bovine submaxillary mucin - TBS Tris-buffered saline - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis - BSA Bovine serum albumin - HA Hemagglutination assay - ALL Acute lymphocytic leukemia - AML Acute myelogenuos leukemia - NP 40 Nonidet 40     

Stereospecific receptors for substance P on cultured human IM-9 lymphoblasts

The neuropeptide substance P (SP), which has been demonstrated to bind specifically to human blood T lymphocytes and to stimulate their uptake of [3H]thymidine and [3H]leucine, now is shown to bind stereospecifically to cultured human lymphoblasts of the IM-9 line. The specific binding of [3H]SP by IM-9 lymphoblasts increases linearly with the concentration of IM-9 lymphoblasts, achieves a plateau after approximately 15 to 20 min at 4 degrees C and 4 to 6 min at 37 degrees C, and is rapidly reversible at both 4 degrees C and 37 degrees C. The binding of [3H]SP at steady-state conditions demonstrates a dissociation constant (KD) of 0.65 +/- 0.19 nM (mean +/- SD, n = 5) and 22,641 +/- 6143 receptors per IM-9 lymphoblast. Maximal specific binding of [3H]SP to IM-9 lymphoblasts is observed at pH 7.4 and is dependent on the presence of Mg2+, but not Ca2+, in the medium. The peptide structural determinants of the inhibition of binding of [3H]SP to IM-9 lymphoblasts by substituent peptides and homologs of SP indicate that the receptors recognize predominantly the carboxy-terminal portion of SP. The characteristics of the interaction of SP with IM-9 lymphoblasts suggests a receptor-directed mechanism by which neuropeptides may modulate specifically the contributions of lymphocytes to immunity.     

Analytical detection of 9(4)-O-acetylated sialoglycoproteins and gangliosides using influenza C virus.

The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O-acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool.     

Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm

To investigate the role of C in the pathogenesis of antisperm antibody (ASA)-mediated infertility, we evaluated the binding and biologic effects of antisperm IgG and autologous C on human sperm. A flow cytometric assay using motile sperm as a target for IgG ASA+ (n = 30) and ASA- (n = 5) sera was developed for the concomitant detection of sperm-bound IgG and the initial (C3d) and terminal (C5b-9) C components on the surface of human sperm. Of the 30 IgG ASA+ sera evaluated by flow cytometry, 15 (50%) and 22 (73.3%) sera were also positive for sperm-bound C3d and C5b-9, respectively. Monomeric IgG purified from C-fixing ASA+ serum was able to bind to sperm and induced deposition of C3 on the sperm surface in the presence of human C. Incubation of motile sperm with C-fixing immune sera resulted in a significant loss (43 to 87%) of motility associated with characteristic C5b-9-induced alterations in sperm morphology leading ultimately to sperm lysis. When motile sperm were cocultured with purified polymorphonuclear leukocytes (PMN) in the presence of C-fixing immune sera, the binding of sperm heads to the PMN resulted in the formation of sperm rosettes, whereas non C-fixing or control sera had no such effect. Transmission electron microscopy of thin sections of the rosettes revealed ingestion of the sperm by the human PMN. These data suggested that 1) antibody bound to sperm is capable of activating autologous C by the classical pathway; 2) binding of both IgG and C proteins initiates C3-mediated sperm binding to PMN and sperm inactivation by deposition of membrane attack complex (MC5b-9) of C; and 3) concomitant detection of sperm-bound IgG, C3d, and C5b-9 may serve as an indicator of C-fixing cytotoxic ASA in the sera of infertile couples.     

Chronic Lymphocytic Leukemia Patients Have a Preserved Cytomegalovirus-Specific Antibody Response despite Progressive Hypogammaglobulinemia

Chronic lymphocytic leukemia (CLL) is characterized by progressive hypogammaglobulinemia predisposing affected patients to a variety of infectious diseases but paradoxically not to cytomegalovirus (CMV) disease. Moreover, we found reactivity of a panel of CLL recombinant antibodies (CLL-rAbs) encoded by a germ-line allele with a single CMV protein, pUL32, despite differing antibody binding motifs. To put these findings into perspective, we studied prospectively relative frequency of viremia, kinetics of total and virus-specific IgG over time, and UL32 genetic variation in a cohort of therapy-naive patients (n=200). CMV-DNA was detected in 3% (6/200) of patients. The decay of total IgG was uniform (mean, 0.03; SD, 0.03) and correlated with that of IgG subclasses 1-4 in the paired samples available (n=64; p<0.001). Total CMV-specific IgG kinetics were more variable (mean, 0,02; SD, 0,06) and mean decay values differed significantly from those of total IgG (p=0.034). Boosts of CMV-specific antibody levels were observed in 49% (22/45) of CMV-seropositive patients. In contrast, VZV- and EBV-specific IgG levels decayed in parallel with total IgG levels (p=0.003 and p=0.001, respectively). VZV-specific IgG even became undetectable in 18% (9/50) of patients whereas CMV-specific ones remained detectable in all seropositive patients. The observed CMV-specific IgG kinetics were predicated upon the highly divergent kinetics of IgG specific for individual antigens - glycoprotein B-specific IgG were boosted in 51% and pUL32-specific IgG in 32% of patients. In conclusion, CLL patients have a preserved CMV-specific antibody response despite progressive decay of total IgG and IgG subclasses. CMV-specific IgG levels are frequently boosted in contrast to that of other herpesviruses indicative of a higher rate of CMV reactivation and antigen-presentation. In contrast to the reactivity of multiple different CLL-rAbs with pUL32, boosts of humoral immunity are triggered apparently by other CMV antigens than pUL32, like glycoprotein B.     

Platelet-activating factor changes in phospholipid extracts from the plasma,peripheral blood mononuclear cells and bone marrow mononuclear cells of patients with acute leukemia — A <Superscript>31</Superscript>P MRS <Emphasis Type="Italic">in vitro</Emphasis> study

The aim of this investigation was to evaluate the changes in PAF concentrations in the plasma, PBMC and BMMC of patients with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The plasma was from 23 healthy volunteers (HV) and 44 patients with AL (16 ALL, 28 AML). The PBMC were from 15 HV and 55 patients with AL (18 ALL, 37 AML), and the BMMC from 40 patients with AL (11 ALL, 29 AML). Methanol-chloroform phospholipid extraction from 60 × 10⁶ cells (PBMC or BMMC) was performed according to a modified version of Folch’s method. ³¹P MRS data was obtained on an AMX 300 Bruker spectrometer (7.05 T). The PAF concentration in the plasma of the patients with ALL or AML was lower than that for the healthy volunteers. The PAF concentration in the plasma of the patients with ALL did not differ significantly from that of the patients with AML. In the case of both the PBMC and BMMC, the PAF concentration was significantly diminished in patients with ALL relative to the concentration for those with AML and for the healthy volunteers. No differences were observed in the PAF concentrations for the AML patients and the healthy volunteers.