Suman Bandyopadhyay

O-acetylation of sialic acids is required for the survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL)

Exploiting the selective affinity of Achatinin-H towards 9-O-acetylneuraminic acid(α2-6)GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on hematopoietic cells of children suffering from acute lymphoblastic leukemia (ALL), indicative of defective sialylation associated with this disease. The carbohydrate epitope of Neu5,9Ac2-GPsALL was confirmed by using several synthetic sialic acid analogues. They are functionally active signaling molecules as demonstrated by their role in mediating lymphoproliferative responses and consequential increased production of IFN-γ due to specific stimulation of Neu5,9Ac2-GPs on PBMCALL with Achatinin-H. Cells devoid of 9-O-acetylations (9-O-AcSA−) revealed decreased nitric oxide production as compared to 9-O-AcSA+ cells on exposure to IFN-γ. Under this condition, a decrease in viability of 9-O-AcSA− cells as compared to 9-O-AcSA+ cells was also observed which was reflected from increased caspase 3 activity and apoptosis suggesting the protective role of this glycotope. These Neu5,9Ac2-GPs are also capable of inducing disease-specific anti-Neu5,9Ac2-GPs antibodies in ALL children. Additionally, we have observed that disease-specific anti-Neu5,9Ac2-GPs have altered glycosylation profile, and they are incapable of exerting a few Fc-glycosylation-sensitive effector functions. These observations hint toward a disbalanced homeostasis, thereby enabling the cancer cells to escape host defense. Taken together, it may be hypothesized that Neu5,9Ac2-GPs and their antibodies play a prominent role in promoting the survival of lymphoblasts in ALL.

Antibodies Directed against O-Acetylated Sialoglycoconjugates Accelerate Complement Activation in Leishmania donovani Promastigotes

BACKGROUND An enhanced presence of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) triggers the alternate pathway (AP) in Indian visceral leishmaniasis (VL). Antibodies directed against these epitopes are present in high titers. The biological relevance of these antibodies, with regard to activation of the classical pathway (CP), was investigated. METHODS Complement activators were affinity purified, complement activation via the CP, AP, and lectin-mediated complement pathway was measured by use of an anti-C3 radio-binding assay, and the number of C3 molecules was quantitated by Scatchard analysis. Cell death induced via the complement pathways was measured by use of MTT (tetrazolium salt 3- [4, 5-dimethylthiazol-2-yl] -2, 5-diphenyltetrazolium bromide) assay, and uptake of propidium iodide (PI) was measured by flow cytometry. RESULTS Anti-O-AcSGs from both healthy donors and patients with VL elicited C3 deposition as early as 3 min, which triggered parasite lysis, as demonstrated by use of MTT assay and corroborated by the high rate of uptake of PI. Analysis of complement activation by mannan-binding lectin and C-reactive protein demonstrated their negligible contribution during the 3-min time frame. CONCLUSIONS Anti-O-AcSGs were identified as an important source of CP activation under normal physiological conditions, suggesting that they play a role in conferring host protection against parasite infection.

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 (PBMCALL, 9‐O‐AcSA+ cells) was demonstrated by using a lectin, Achatinin‐H, whose lectinogenic epitope was 9‐O‐AcSAα2‐6GalNAc. Our aim was to evaluate the in vitro contributory role of this glycotope (9‐O‐AcSAα2‐6GalNAc) 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 PBMCALL using O‐acetyl esterase. An elevated level of serum interferon gamma (IFN‐γ) in affected children led us to think that PBMCALL are continuously exposed specifically to this cytokine. Accordingly, 9‐O‐AcSA+ and 9‐O‐AcSA− cells were exposed in vitro to IFN‐γ. 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‐γ 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 PBMCALL. Taken together, it is reasonable to hypothesise that O‐acetylation of sialic acids on PBMCALL may be an additional mechanism that promotes the survival of lymphoblasts by avoiding apoptosis via IFN‐γ‐induced NO production.

Increased interferon gamma production by peripheral blood mononuclear cells in response to stimulation of overexpressed disease-specific 9-O-acetylated sialoglycoconjugates in children suffering from acute lymphoblastic leukaemia

Disease‐specific over‐expression of 9‐O‐acetylated sialoglycoconjugates (9‐O‐AcSGs) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia (ALL, PBMCALL) has been demonstrated using a lectin, Achatinin‐H, with specificity towards 9‐O‐AcSAα2‐6GalNAc. This study investigated the contributory role of 9‐O‐AcSGs induced on PBMCALL. Stimulation of PBMCALL with Achatinin‐H through 9‐O‐AcSGs led to a lymphoproliferative response with a significantly increased interferon‐γ (IFN‐γ) production when compared with unstimulated cells as demonstrated by enzyme‐linked immunosorbent assay and mRNA expression. Under identical conditions, PBMCALL ablated of O‐acetylations did not respond to such stimulation. In summary, it may be concluded that stimulation of over‐expressed 9‐O‐AcSGs regulate signalling for proliferation, leading to the release of IFN‐γ. Controlled expression of these molecules may be exploited as potential targets for therapy, promising beneficial effects to children with ALL.

Flow-cytometric monitoring of disease-associated expression of 9-O-acetylated sialoglycoproteins in combination with known CD antigens, as an index for MRD in children with acute lymphoblastic leukaemia: a two-year longitudinal follow-up study

BackgroundOver expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs, abbreviated as OAcSGP) has been demonstrated as a disease-associated antigen on the lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Achatinin-H, a lectin, has selective affinity towards terminal 9-O-acetylated sialic acids-α2-6-Nacetylated galactosamine. Exploring this affinity, enhanced expression of OAcSGP was observed, at the onset of disease, followed by its decrease with chemotherapy and reappearance with relapse. In spite of treatment, patients retain the diseased cells referred to as minimal residual disease (MRD) responsible for relapse. Our aim was to select a suitable template by using the differential expression of OAcSGP along with other known CD antigens to monitor MRD in peripheral blood (PB) and bone marrow (BM) of Indian patients with B- or T-ALL during treatment and correlate it with the disease status.MethodsA two-year longitudinal follow-up study was done with 109 patients from the onset of the disease till the end of chemotherapy, treated under MCP841protocol. Paired samples of PB (n = 1667) and BM (n = 999) were monitored by flow cytometry. Three templates selected for this investigation were OAcSGP+CD10+CD19+ or OAcSGP+CD34+CD19+ for B-ALL and OAcSGP+CD7+CD3+ for T-ALL.ResultsUsing each template the level of MRD detection reached 0.01% for a patient in clinical remission (CR). 81.65% of the patients were in CR during these two years while the remaining relapsed. Failure in early clearance of lymphoblasts, as indicated by higher MRD, implied an elevated risk of relapse. Soaring MRD during the chemotherapeutic regimen predicted clinical relapse, at least a month before medical manifestation. Irrespective of B- or T-lineage ALL, the MRD in PB and BM correlated well.ConclusionA range of MRD values can be predicted for the patients in CR, irrespective of their lineage, being 0.03 ± 0.01% (PB) and 0.05 ± 0.015% (BM). These patients may not be stated as normal with respect to the presence of MRD. Hence, MRD study beyond two-years follow-up is necessary to investigate further reduction in MRD, thereby ensuring their disease-free survival. Therefore, we suggest use of these templates for MRD detection, during and post-chemotherapy for proper patient management strategies, thereby helping in personalizing the treatment.

Co-expression of 9-O-acetylated sialoglycoproteins and their binding proteins on lymphoblasts of childhood acute lymphoblastic leukemia: an anti-apoptotic role

Abstract Enhanced levels of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2GPs) as disease-associated molecules was reported to act as signaling molecules for promoting survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL). Here, we searched for potential physiological ligands for Neu5,9Ac2GPs that could be involved in modulating the survival of lymphoblasts. Accordingly, we examined the presence of binding proteins for Neu5,9Ac2GPs on cell lines and primary cells of patients with B- and T-ALL, at presentation of the disease. Peripheral blood mononuclear cells from normal healthy donors and cells from myeloid leukemia patients were used for comparison. Neu5,9Ac2GPs-binding proteins (BPs) were specifically detected on the surface of both T- and B-ALL-lymphoblasts and ALL-cell lines along with the consistent presence of Neu5,9Ac2GPs. The Neu5,9Ac2GPs and BPs also co-localized on the cell surface and interacted specifically in vitro. Apoptosis of lymphoblasts, induced by serum starvation, was reversed in the presence of purified Neu5,9Ac2GPs due to possible engagement of BPs, and the anti-apoptotic role of this interaction was established. This is the first report of the presence of potential physiological ligands for disease-associated molecules like Neu5,9Ac2GPs, the interaction of which is able to trigger an anti-apoptotic signal conferring a survival advantage to leukemic cells in childhood ALL.